Stabilisation and isolation of extracellular nucleic acids

ABSTRACT

The present invention provides methods, compositions and devices for stabilizing the extracellular nucleic acid population in a cell-containing biological sample using an apoptosis inhibitor, preferably a caspase inhibitor, a hypertonic agent and/or a compound according to formula (1) as defined in the claims.

The work leading to this invention has received funding from theEuropean Community's Seventh Framework Programme (FP7/2007-2013) undergrant agreement no 222916.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 760204_401USPC_SEQUENCE_LISTING.txt. The textfile is 22.4 KB, was created on Mar. 24, 2014, and is being submittedelectronically via EFS-Web.

FIELD OF THE INVENTION

The technology disclosed herein relates to methods and compositionssuitable for stabilizing the extracellular nucleic acid population in acell-containing sample, in particular a blood sample, and to a methodfor isolating extracellular nucleic acids from respectively stabilizedbiological samples.

BACKGROUND

Extracellular nucleic acids have been identified in blood, plasma, serumand other body fluids. Extracellular nucleic acids that are found inrespective samples are to a certain extent degradation resistant due tothe fact that they are protected from nucleases (e.g. because they aresecreted in form of a proteolipid complex, are associated with proteinsor are contained in vesicles). The presence of elevated levels ofextracellular nucleic acids such as DNA and/or RNA in many medicalconditions, malignancies, and infectious processes is of interest interalia for screening, diagnosis, prognosis, surveillance for diseaseprogression, for identifying potential therapeutic targets, and formonitoring treatment response. Additionally, elevated fetal DNA/RNA inmaternal blood is being used to determine e.g. gender identity, assesschromosomal abnormalities, and monitor pregnancy-associatedcomplications. Thus, extracellular nucleic acids are in particularuseful in non-invasive diagnosis and prognosis and can be used e.g. asdiagnostic markers in many fields of application, such as non-invasiveprenatal genetic testing, oncology, transplantation medicine or manyother diseases and, hence, are of diagnostic relevance (e.g. fetal- ortumor-derived nucleic acids). However, extracellular nucleic acids arealso found in healthy human beings. Common applications and analysismethods of extracellular nucleic acids are e.g. described inWO97/035589, WO97/34015, Swarup et al, FEBS Letters 581 (2007) 795-799,Fleischhacker Ann. N.Y. Acad. Sci. 1075: 40-49 (2006), Fleischhacker andSchmidt, Biochmica et Biophysica Acta 1775 (2007) 191-232, Hromadnikovaet al (2006) DNA and Cell biology, Volume 25, Number 11 pp 635-640; Fanet al (2010) Clinical Chemistry 56:8.

Traditionally, the first step of isolating extracellular nucleic acidsfrom a cell-containing biological sample such as blood is to obtain anessentially cell-free fraction of said sample, e.g. either serum orplasma in the case of blood. The extracellular nucleic acids are thenisolated from said cell-free fraction, commonly plasma, when processinga blood sample. However, obtaining an essentially cell-free fraction ofa sample can be problematic and the separation is frequently a tediousand time consuming multi-step process as it is important to usecarefully controlled conditions to prevent cell breakage duringcentrifugation which could contaminate the extracellular nucleic acidswith cellular nucleic acids released during breakage. Furthermore, it isoften difficult to remove all cells. Thus, many processed samples thatare often and commonly classified as “cell-free” such as plasma or serumin fact still contain residual amounts of cells that were not removedduring the separation process. Another important consideration is thatcellular nucleic acid are released from the cells contained in thesample due to cell breakage during ex vivo incubation, typically withina relatively short period of time from a blood draw event. Once celllysis begins, the lysed cells release additional nucleic acids whichbecome mixed with the extracellular nucleic acids and it becomesincreasingly difficult to recover the extracellular nucleic acids fortesting. These problems are discussed in the prior art (see e.g. Chiu etal (2001), Clinical Chemistry 47:9 1607-1613; Fan et al (2010) andUS2010/0184069). Further, the amount and recoverability of availableextracellular nucleic acids can decrease substantially over a period oftime due to degradation.

Besides mammalian extracellular nucleic acids that derive e.g. fromtumor cells or the fetus, cell-containing samples may also compriseother nucleic acids of interest that are not comprised in cells. Animportant, non-limiting example is pathogen nucleic acids such as viralnucleic acids. Preservation of the integrity of viral nucleic acids incell-containing samples such as in particular in blood specimens duringshipping and handling is also crucial for the subsequent analysis andviral load monitoring.

The above discussed problems particularly are an issue, if the samplecomprises a high amount of cells as is the case e.g. with whole bloodsamples. Thus, in order to avoid respectively reduce the above describedproblems it is common to separate an essentially cell-free fraction ofthe sample from the cells contained in the sample basically immediatelyafter the sample is obtained. E.g. it is recommended to obtain bloodplasma from whole blood basically directly after the blood is drawnand/or to cool the whole blood and/or the obtained plasma or serum inorder to preserve the integrity of the extracellular nucleic acids andto avoid contaminations of the extracellular nucleic acid populationwith intracellular nucleic acids that are released from the containedcells. However, the need to directly separate e.g. the plasma from theblood is a major disadvantage because many facilities wherein the bloodis drawn (e.g. a doctor's practice) do not have a centrifuge that wouldenable the efficient separation of blood plasma. Furthermore, plasmathat is obtained under regular conditions often comprises residualamounts of cells which accordingly, may also become damaged or may dieduring handling of the sample, thereby releasing intracellular nucleicacids, in particular genomic DNA, as is described above. These remainingcells also pose a risk that they become damaged during the handling sothat their nucleic acid content, particularly genomic (nuclear) DNA andcytoplasmic RNA, would merge with and thereby contaminate respectivelydilute the extracellular, circulating nucleic acid fraction. To removethese remaining contaminating cells and to avoid/reduce theaforementioned problems, it was known to perform a second centrifugationstep at higher speed. However, again, such powerful centrifuges areoften not available at the facilities wherein the blood is obtained.Furthermore, even if plasma is obtained directly after the blood isdrawn, it is recommended to freeze it at −80° C. in order to preservethe nucleic acids contained therein if the nucleic acids can not bedirectly isolated. This too imposes practical constraints upon theprocessing of the samples as e.g. the plasma samples must be shippedfrozen. This increases the costs and furthermore, poses a risk that thesample gets compromised in case the cold chain is interrupted.

Blood samples are presently usually collected in blood collection tubescontaining spray-dried or liquid EDTA (e.g. BD Vacutainer K₂EDTA). EDTAchelates magnesium, calcium and other bivalent metal ions, therebyinhibiting enzymatic reactions, such as e.g. blood clotting or DNAdegradation due to DNases. However, even though EDTA is an efficientanticoagulant, EDTA does not efficiently prevent the dilutionrespectively contamination of the extracellular nucleic acid populationby released intracellular nucleic acids. Thus, the extracellular nucleicacid population that is found in the cell-free portion of the samplechanges during the storage. Accordingly, EDTA is not capable ofsufficiently stabilising the extracellular nucleic acid population inparticular because it can not avoid the contamination of theextracellular nucleic acid population with e.g. genomic DNA fragmentswhich are generated after blood draw by cell degradation and cellinstability during sample transportation and storage.

Methods are known in the prior art that specifically aim at stabilizingcirculating nucleic acids contained in whole blood. One method employsthe use of formaldehyde to stabilize the cell membranes, therebyreducing the cell lysis and furthermore, formaldehyde inhibitsnucleases. Respective methods are e.g. described in U.S. Pat. Nos.7,332,277 and 7,442,506. However, the use of formaldehyde orformaldehyde-releasing substances has drawbacks, as they may compromisethe efficacy of extracellular nucleic acid isolation by induction ofcrosslinks between nucleic acid molecules or between proteins andnucleic acids. Alternative methods to stabilize blood samples aredescribed e.g. in US 2010/0184069 and US 2010/0209930. These ratherrecently developed methods demonstrate the great need for providingmeans to stabilise cell-containing biological samples, to allow theefficient recovery of e.g. extracellular nucleic acids contained in suchsamples.

However, despite these rather recent developments there is still acontinuous need to develop sample processing techniques which result ina stabilisation of the extracellular nucleic acid population comprisedin a biological sample, in particular a sample containing cells,including samples suspected of containing cells, in particular wholeblood, plasma or serum, thereby making the handling, respectivelyprocessing of such samples easier (e.g. by avoiding the need to directlyseparate plasma from whole blood or to cool or even freeze the isolatedplasma) thereby also making the isolation and testing of extracellularnucleic acids contained in such samples more reliable and consequently,thereby improving the diagnostic and prognostic capabilities of theextracellular nucleic acids. In particular, there is a continuous needfor a solution for preserving extracellular nucleic acids in whole bloodsamples, e.g. for prenatal testing and/or for screening for neoplastic,in particular premalignant or malignant diseases.

It is the object of the present invention to overcome at least one ofthe drawbacks of the prior art sample stabilization methods. Thus, it isinter alia an object of the present invention to provide a method thatis capable of stabilising a cell-containing sample, in particular wholeblood. In particular, it is an object of the present invention tostabilise the extracellular nucleic acid population contained in abiological sample and in particular to avoid a contamination of theextracellular nucleic acid population with genomic DNA, in particularfragmented genomic DNA. Furthermore, it is in particular an object ofthe present invention to provide a method tsuitable for stabilising abiological sample, preferably a whole blood sample, even at roomtemperature, preferably for a period of at least two, preferably atleast three days. Furthermore, it is an object of the present inventionto provide a sample collection container, in particular a bloodcollection tube that is capable of effectively stabilising a biologicalsample and in particular the extracellular nucleic acid populationcomprised in the sample.

SUMMARY OF THE INVENTION

The present invention is based on the finding that certain additives aresurprisingly effective in stabilizing cell-containing biological samplescomprising extracellular nucleic acids, in particular whole bloodsamples or samples derived from whole blood such as e.g. blood plasma.It was found that these additives are highly efficient in stabilizingthe extracellular nucleic acid population and in particular are capableto avoid or at least significantly reduce contaminations with genomicDNA, in particular fragmented genomic DNA.

According to a first aspect, a method suitable for stabilizing anextracellular nucleic acid population comprised in a cell-containingsample is provided, wherein a sample is contacted with

-   -   a) at least one apoptosis inhibitor,    -   b) at least one hypertonic agent, which stabilizes the cells        comprised in the sample, and/or    -   c) at least one compound according to formula 1

wherein R1 is a hydrogen residue or an alkyl residue, preferably a C1-C5alkyl residue, more preferred a methyl residue, R2 and R3 are identicalor different hydrocarbon residues with a length of the carbon chain of1-20 atoms arranged in a linear or branched manner, and R4 is an oxygen,sulphur or selenium residue.

According to a first sub-aspect, a method suitable for stabilizing anextracellular nucleic acid population comprised in a cell-containingsample is provided, wherein the sample is contacted with at least oneapoptosis inhibitor. Preferably, the cell-containing sample is selectedfrom whole blood, plasma or serum. Surprisingly, it was found that theapoptosis inhibitor reduces contaminations of the extracellular nucleicacid population with intracellular nucleic acids, in particularfragmented genomic DNA, that originate from cells contained in thesample, e.g. from damaged or dying cells. Furthermore, the inventorsfound that the apoptosis inhibitor reduces the degradation of nucleicacids present in the sample. Thus, the stabilization according to thepresent invention using an apoptosis inhibitor has the effect that theextracellular nucleic acid population contained in the sample issubstantially preserved in the state it had shown at the time thebiological sample was obtained, respectively collected.

According to a second sub-aspect, a method suitable for stabilizing anextracellular nucleic acid population comprised in a cell-containingsample is provided, wherein a sample is contacted with at least onehypertonic agent, which is capable of stabilizing cells comprised in thesample. It was surprisingly found that cell shrinking that is induced bymild hypertonic effects (osmosis) results in a considerable increase ofthe cell stability. By increasing the cell stability, the hypertonicagent in particular reduces the release of intracellular nucleic acids,in particular genomic DNA, from the contained cells into theextracellular portion or compartment of the sample. Thus, thestabilization according to the present invention using a hypertonicagent has the effect that the extracellular nucleic acid populationcontained in the sample is substantially preserved in the state it hadshown at the time the biological sample was obtained, respectivelycollected.

According to a third sub-aspect of the present invention, a methodsuitable for stabilizing an extracellular nucleic acid populationcomprised in a cell-containing sample is provided, wherein a sample iscontacted with at least one compound according to formula 1

wherein R1 is a hydrogen residue or an alkyl residue, preferably a C1-C5alkyl residue, more preferred a methyl residue, R2 and R3 are identicalor different hydrocarbon residues with a length of the carbon chain of1-20 atoms arranged in a linear or branched manner, and R4 is an oxygen,sulphur or selenium residue. It was found that adding a respectivecompound as an advantageous stabilizing effect on the extracellularnucleic acid population.

According to a fourth sub-aspect, a method suitable for stabilizing anextracellular nucleic acid population comprised in a cell-containingsample is provided, wherein a sample is contacted with

-   -   a) at least one apoptosis inhibitor, and    -   b) at least one hypertonic agent, which stabilizes the cells        comprised in the sample.

It was found that the combination of these stabilizing agents (andoptionally further additives) is remarkably effective in inhibiting therelease of intracellular nucleic acids, in particular genomic DNA, fromthe contained cells into the extracellular portion of the sample.Furthermore, it was shown that the degradation of nucleic acids presentin the sample is highly efficiently prevented. In particular, lessfragmented genomic DNA is found in respectively stabilized samples.Thus, the stabilization according to the present invention using thiscombination of stabilizing additives has the effect that theextracellular nucleic acid population contained in the sample issubstantially and effectively preserved in the state it had shown at thetime the biological sample was obtained, respectively collected (e.g.drawn in the case of blood) and that in particular contaminations of theextracellular nucleic acid population with fragmented genomic DNA arereduced.

In order to enhance the stabilization effect towards extracellularnucleic acids, it is also an object of the present invention to providefurther combinations of stabilizing agents in order to stabilize theextracellular nucleic acid population comprised in a cell-containingsample. A respective combination may comprise at least one apoptosisinhibitor, at least one hypertonic agent and/or at least one compoundaccording to formula 1 as defined above, for example (1) a combinationof at least one apoptosis inhibitor and at least one compound accordingto formula 1 as defined above, (2) a combination of at least onehypertonic agent and at least one compound according to formula 1 or (3)a combination of all three stabilizing agents, i.e. at least oneapoptosis inhibitor, at least one hypertonic agent and at least onecompound according to formula 1. A respective combination may alsocomprise additional additives that enhance the stabilizing effect suchas e.g. chelating agents. In case the sample is blood or a samplederived from blood, usually an anticoagulant is also added. Chelatingagents such as e.g. EDTA are suitable for this purpose. Respectivestabilizing combinations can be according to a fifth sub-aspectadvantageously used in a method suitable for stabilizing anextracellular nucleic acid population comprised in a cell-containingsample according to the first aspect of the present invention.

According to a second aspect, a method for isolating extracellularnucleic acids from a biological sample is provided, wherein said methodcomprises the steps of:

-   a) stabilizing the extracellular nucleic acid population comprised    in a sample according to the method defined in the first aspect of    the present invention; and-   b) isolating extracellular nucleic acids from said sample.

Stabilization in step a) can be achieved e.g. according to one of thefive sub-aspects of the first aspect according to the present inventionas described above. As discussed above, the stabilization according tothe present invention has the effect that the extracellular nucleic acidpopulation contained in the sample is substantially preserved in thestate it had shown at the time the biological sample was obtained,respectively collected. Therefore, extracellular nucleic acids obtainedfrom a respectively stabilized sample comprise less contaminations withintracellular nucleic acids, in particular fragmented genomic DNA, thatresults e.g. from decaying cells comprised in the sample compared toextracellular nucleic acids that are obtained from an unstabilizedsample. The substantial preservation of the extracellular nucleic acidpopulation is an important advantage because thisstabilization/preservation enhances the accuracy of any subsequenttests. It allows for standardizing the isolation and subsequent analysisof the extracellular nucleic acid population, thereby making diagnosticor prognostic applications that are based on the extracellular nucleicacid fraction more reliable and more independent from the usedstorage/handling conditions. Thereby, the diagnostic and prognosticapplicability of the respectively isolated extracellular nucleic acidsis improved. In particular, the teachings of the present invention havethe advantage that the ratio of certain extracellular nucleic acidmolecules can be kept substantially constant compared to the ratio atthe time the sample was collected. The stabilization achieves thatintracellular nucleic acids are substantially kept within the cells andthat extracellular nucleic acids are substantially stabilized.

According to a third aspect, a composition suitable for stabilizing acell-containing biological sample is provided, comprising:

-   -   a) at least one apoptosis inhibitor, preferably a caspase        inhibitor, and/or    -   b) at least one hypertonic agent which is suitable for        stabilizing the cells comprised in the sample, preferably a        hydroxylated organic compound; and/or    -   c) at least one compound according to formula 1 as defined        above; and/or    -   d) optionally at least one anticoagulant, preferably a chelating        agent.

A respective stabilizing composition is particularly effective instabilizing a cell-containing biological sample, in particular wholeblood, plasma and/or serum by stabilizing the cells and theextracellular nucleic acid population comprised in said sample.Preferably, at least two of the stabilizing agents defined in a) to c)more preferred all of the stabilizing agents defined in a) to c) arepresent in the stabilizing composition. A respective stabilizingcomposition allows the storage and/or handling, e.g. shipping, of thesample, e.g. whole blood, at room temperature for at least two, orpreferably at least three days without substantially compromising thequality of the sample, respectively the extracellular nucleic acidpopulation contained therein. Thus, when using the stabilizationcomposition according to the present invention, the time between samplecollection, e.g. blood collection, and nucleic acid extraction can varywithout substantial effect on the extracellular nucleic acid populationcontained in the sample. This is an important advantage as it reducesthe variability in the extracellular nucleic acid populationattributable to different handling procedures.

According to a forth aspect, a container for collecting acell-containing biological sample, preferably a blood sample, isprovided wherein the container comprises a composition according to thethird aspect of the present invention. Providing a respective container,e.g. a sample collection tube comprising the stabilizing composition hasthe advantage that the sample is immediately stabilized as soon as thesample is collected in the respective container. Furthermore, arespective sample collection container, in particular a blood collectiontube, is capable of stabilising blood cells and extracellular nucleicacids and optionally, viruses respectively viral nucleic acids containedin a blood sample or a sample derived from blood. Thereby, a furtherproblem was overcome.

According to a fifth aspect, a method is provided comprising the step ofcollecting, preferably withdrawing, a biological sample, preferablyblood, from a patient directly into a chamber of a container accordingto the fourth aspect of the present invention.

According to a sixth aspect, a method of producing a compositionaccording to the third aspect of the present invention is provided,wherein the components of the composition are mixed, preferably aremixed in a solution. The term “solution” as used herein in particularrefers to a liquid composition, preferably an aqueous composition. Itmay be a homogenous mixture of only one phase but it is also within thescope of the present invention that a solution comprises solidcomponents such as e.g. precipitates.

Other objects, features, advantages and aspects of the presentapplication will become apparent to those skilled in the art from thefollowing description and appended claims. It should be understood,however, that the following description, appended claims, and specificexamples, while indicating preferred embodiments of the application, aregiven by way of illustration only. Various changes and modificationswithin the spirit and scope of the disclosed invention will becomereadily apparent to those skilled in the art from reading the following.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a shows a gel picture after chip electrophoresis of DNA isolatedfrom samples treated with caspase inhibitors (Example 1).

FIG. 1b is a diagram showing the effect of caspase inhibitors on theincrease of ribosomal 18S DNA in plasma (Example 1).

FIG. 2a shows a gel picture after chip electrophoresis of DNA isolatedfrom samples treated with different concentrations of the caspaseinhibitor Q-VD-OPH in combination (Example 2).

FIG. 2b is a diagram showing the effects of different concentrations ofthe caspase-inhibitor Q-VD-OPH in combination with glucose on theincrease of ribosomal 18S DNA in the plasma (Example 2).

FIG. 3 shows the blood cell integrity measured by flow cytometry forblood cells treated with dihydroxyacetone dissolved in different buffers(Example 3).

FIG. 4a shows a gel picture after chip electrophoresis of DNA isolatedfrom samples treated with dihydroxyacetone dissolved in differentbuffers (Example 3).

FIG. 4b is a diagram showing the effect of dihydroxyacetone on theincrease of ribosomal 18S DNA (Example 3).

FIG. 5 shows the blood cell integrity measured by flow cytometry forblood cells treated with different concentrations of dihydroxyacetone(Example 4).

FIG. 6a shows a gel picture after chip electrophoresis of DNA isolatedfrom samples treated with different concentrations of dihydroxyacetone(Example 4).

FIG. 6b is a diagram showing the effect of different dihydroxyacetoneconcentrations on the increase of ribosomal 18S DNA (Example 4).

FIG. 7a shows the blood cell integrity measured by flow cytometry forblood cells treated with a combination of elevated K₂EDTA, Q-VD-OPH andDHA (Example 5).

FIG. 7b is a diagram showing the effect of the combination of EDTA, DHAand Q-VD-OPH on the increase of 18S DNA (Example 5).

FIG. 8 is a diagram showing the effect of the combination of EDTA, DHAand Q-VD-OPH on the transcript level of free circulating mRNA in plasma(Example 6).

FIG. 9 is a diagram showing the effects of different concentrations ofDMAA on the increase of ribosomal 18S DNA in the plasma.

FIG. 10 is a diagram showing the influence of different sugar alcoholson the increase of 18S rDNA (Example 8)

FIG. 11 is a diagram showing the influence of substances on the increaseof 18S rDNA (Example 9)

FIG. 12 is a diagram showing the influence of substances on the increaseof 18S rDNA (Example 10)

FIG. 13 is a diagram showing the influence of substances on the increaseof 18S rDNA (Example 11)

FIG. 14 is a diagram showing the influence of substances on the increaseof 18S rDNA (Example 11)

FIG. 15 is a diagram showing the influence of substances on the increaseof 18S rDNA

FIG. 16 is a diagram showing the ccfDNA increase in plasma fraction ofwhole blood incubated for up to 6 days at 37° C. (Example 13)

FIG. 17 is a diagram showing the ccfDNA increase in plasma fraction ofwhole blood incubated for up to 6 days at 37° C. (Example 13)

FIG. 18 is a diagram showing the percent hits of spiked-in DNA fragments(Example 14)

FIG. 19 is a diagram showing the mean copies (Example 14)

FIG. 20 is a diagram showing the percent of 18S compared to BDVacutainer K2E (Example 14)

FIG. 21 is a diagram showing the decrease of HIV, incubated in wholeblood at 37° C., purified from plasma (Example 15)

FIG. 22 is a diagram showing the decrease of HCV, incubated in wholeblood at 37° C., purified from plasma (Example 15)

FIG. 23 is a diagram showing the influence of propionamid on 18S rDNAincrease Donor 1 (Example 16)

FIG. 24 is a diagram showing the influence of propionamid on 18S rDNAincrease Donor 2 (Example 16)

DETAILED DESCRIPTION OF THIS INVENTION

The present invention is directed to methods, compositions and devicesand thus to technologies suitable for stabilizing the extracellularnucleic acid population comprised in a cell-containing biologicalsample. The stabilization technologies disclosed herein reduce the riskthat the extracellular nucleic acid population is contaminated withintracellular nucleic acids, in particular fragmented genomic DNA, whichderives from, e.g. is released from damaged and/or dying cells containedin the sample. Therefore, the present invention achieves thestabilization of the sample and hence the stabilization of theextracellular nucleic acid population comprised therein without thelysis of the contained cells. Rather, cells contained in the sample arestabilized thereby substantially preventing or reducing the release ofintracellular nucleic acids. The remarkable stabilization that isachieved with the methods and compositions of the present inventionallows the storage and/or handling of the stabilized sample for aprolonged period of time at room temperature without jeopardizing thequality of the sample, respectively the extracellular nucleic acidscontained therein. As the composition of the extracellular nucleic acidpopulation is stabilized and thus substantially preserved at the timethe sample is obtained by using the teachings of the present invention,the time between sample collection and nucleic acid extraction can varywithout significant effect on the composition of the extracellularnucleic acids population. This allows the standardization of e.g.diagnostic or prognostic extracellular nucleic acid analysis becausevariations in the handling/storage of the samples have less influence onthe quality, respectively the composition of the extracellular nucleicacid population, thereby providing an important advantage over prior artmethods. Hence, the samples, respectively the extracellular nucleicacids obtained from respectively stabilized samples become morecomparable. Furthermore, the teachings of the present invention obviatethe necessity to directly separate cells contained in the sample fromthe cell-free portion of the sample in order to avoid, respectivelyreduce contaminations of the extracellular nucleic acids withintracellular nucleic acids, in particular fragmented genomic DNA, thatis otherwise released from decaying cells. This advantage considerablysimplifies the handling of the samples, in particular the handling ofwhole blood samples. E.g. whole blood samples obtained in a clinic andstabilized according to the teachings of the present invention can beshipped at room temperature and the plasma containing the extracellularnucleic acids can be conveniently separated in the receiving clinicallab. However, the teachings of the invention are also advantageous whenprocessing cell-depleted biological samples, or samples commonlyreferred to as “cell-free” such as e.g. blood plasma or serum.Respective cell-depleted or “cell-free” biological samples may still(also depending on the used separation process) comprise residual cells,in particular white blood cells which comprise genomic DNA, whichaccordingly, pose a risk that the extracellular nucleic acid populationbecomes increasingly contaminated with intracellular nucleic acids, inparticular fragmented genomic DNA, if the (potentially) remaining cellsare damaged or die during the shipping of storing process. This risk isconsiderably reduced when using the stabilization method taught by thepresent invention. Because the technology of the present inventionallows to efficiently preserve the extracellular nucleic acid populationof the sample at the time the sample is collected and contacted with thestabilizing agents, said samples can be properly worked up in thereceiving facilities in order to isolate the extracellular nucleic acidsfrom said samples while substantially avoiding respectively reducingcontaminations of the extracellular nucleic population withintracellular nucleic acids. The facilities receiving the samples suchas e.g. laboratories usually also have the necessary equipment such ase.g. high speed centrifuges (or other means, see also below) toefficiently remove cells comprised in the samples, including residualcells that might be present in cell-depleted samples such as e.g. inblood plasma. Such equipment is often not present in the facilitieswhere the sample is obtained. Thus, the present invention has manyadvantages when stabilizing biological samples which comprise a largeamount of cells such as e.g. whole blood samples, but also has importantadvantages when stabilizing biological samples which comprise only asmall amount of cells or which may only be suspected of containing cellssuch as e.g. plasma, serum, urine, saliva, synovial fluids, amnioticfluid, lachrymal fluid, ichors, lymphatic fluid, liquor, cerebrospinalfluid and the like.

According to a first aspect, a method suitable for stabilizing theextracellular nucleic acid population comprised in a cell-containingsample, preferably a blood sample, is provided, by contacting the samplewith

-   -   a) at least one apoptosis inhibitor, and/or    -   b) at least one hypertonic agent, which stabilizes the cells        comprised in the sample, and/or    -   c) at least one compound according to formula 1

-   -   wherein R1 is a hydrogen residue or an alkyl residue, preferably        a C1-C5 alkyl residue, more preferred a methyl residue, R2 and        R3 are identical or different hydrocarbon residues with a length        of the carbon chain of 1-20 atoms arranged in a linear or        branched manner, and R4 is an oxygen, sulphur or selenium        residue.

Thereby, the risk is reduced that the extracellular nucleic acidpopulation is contaminated with intracellular nucleic acids, inparticular fragmented genomic DNA originating from contained cells, e.g.from damaged or dying cells and/or the degradation of nucleic acidspresent in the sample is reduced, respectively inhibited. This has theeffect that the composition of the extracellular nucleic acid populationcomprised in said sample is substantially preserved, respectivelystabilized.

The term “extracellular nucleic acids” or “extracellular nucleic acid”as used herein, in particular refers to nucleic acids that are notcontained in cells. Respective extracellular nucleic acids are alsooften referred to as cell-free nucleic acids. These terms are used assynonyms herein. Hence, extracellular nucleic acids usually are presentexterior of a cell or exterior of a plurality of cells within a sample.The term “extracellular nucleic acids” refers e.g. to extracellular RNAas well as to extracellular DNA. Examples of typical extracellularnucleic acids that are found in the cell-free fraction (respectivelyportion) of biological samples such as body fluids such as e.g. bloodplasma include but are not limited to mammalian extracellular nucleicacids such as e.g. extracellular tumor-associated or tumor-derived DNAand/or RNA, other extracellular disease-related DNA and/or RNA,epigenetically modified DNA, fetal DNA and/or RNA, small interfering RNAsuch as e.g. miRNA and siRNA, and non-mammalian extracellular nucleicacids such as e.g. viral nucleic acids, pathogen nucleic acids releasedinto the extracellular nucleic acid population e.g. from prokaryotes(e.g. bacteria), viruses, eukaryotic parasites or fungi. According toone embodiment, the extracellular nucleic acid is obtained fromrespectively is comprised in a body fluid as cell-containing biologicalsample such as e.g. blood, plasma, serum, saliva, urine, liquor,cerebrospinal fluid, sputum, lachrymal fluid, sweat, amniotic orlymphatic fluid. Herein, we refer to extracellular nucleic acids thatare obtained from circulating body fluids as circulating extracellularor circulating cell-free nucleic acids. According to one embodiment, theterm extracellular nucleic acid in particular refers to mammalianextracellular nucleic acids, preferably disease-associated ordisease-derived extracellular nucleic acids such as tumor-associated ortumor-derived extracellular nucleic acids, extracellular nucleic acidsreleased due to inflammations or injuries, in particular traumata,extracellular nucleic acids related to and/or released due to otherdiseases, or extracellular nucleic acids derived from a fetus. The term“extracellular nucleic acids” or “extracellular nucleic acid” asdescribed herein also refers to extracellular nucleic acids obtainedfrom other samples, in particular biological samples other than bodyfluids. Usually, more than one extracellular nucleic acid is comprisedin a sample. Usually, a sample comprises more than one kind or type ofextracellular nucleic acids. The term “extracellular nucleic acidpopulation” as used herein in particular refers to the collective ofdifferent extracellular nucleic acids that are comprised in acell-containing sample. A cell-containing sample usually comprises acharacteristic and thus unique extracellular nucleic acid population.Thus, the type, kind and/or the amount of one or more extracellularnucleic acids comprised in the extracellular nucleic acid population ofa specific sample are important sample characteristics. As discussedabove, it is therefore important to stabilize and thus to substantiallypreserve said extracellular nucleic acid population as its compositionand/or the amount of one or more extracellular nucleic acids comprisedin the extracellular nucleic acid population of a sample, can providevaluable information in the medical, prognostic or diagnostic field. Inparticular, it is important to reduce the contamination and hencedilution of the extracellular nucleic acid population by intracellularnucleic acids, in particular by genomic DNA, after the sample wascollected. The substantial preservation of the extracellular nucleicacid population that can be achieved with the stabilization technologiesaccording to the invention allows the population of extracellularnucleic acids within a sample to be maintained substantially unchangedover the stabilization period as compared to the population ofextracellular nucleic acids at the moment of sample stabilization. Atleast, changes in the extracellular nucleic acid population with respectto the quantity, the quality and/or the composition of the comprisedextracellular nucleic acids, in particular changes attributable to anincrease of released genomic DNA, are over the stabilization periodconsiderably reduced (preferably by at least 60%, at least 70%, at least75%, at least 80%, at least 85%, at least 90% or at least 95%) comparedto an unstabilized sample or a corresponding sample that is e.g.stabilized by EDTA in case of a blood sample or a sample derived fromblood.

According to a first sub-aspect of the first aspect, at least oneapoptosis inhibitor is used for stabilizing the sample. As is shown bythe provided examples, already the apoptosis inhibitor alone iseffective in stabilizing a cell-containing sample and to substantiallypreserve the extracellular nucleic acid population from changes in itscomposition in particular arising from contaminations with fragmentedgenomic DNA. The sample can be contacted with the apoptosis inhibitor,e.g. by adding the apoptosis inhibitor to the sample or vice versa. Theat least one apoptosis inhibitor present in the resulting mixturesupports the stabilization of cells contained in the sample and inhibitsthe degradation of nucleic acids comprised in the sample therebysubstantially preserving the extracellular nucleic acid population.

The term “apoptosis inhibitor” as used herein in particular refers to acompound whose presence in a cell-containing biological sample providesa reduction, prevention and/or inhibition of apoptotic processes in thecells and/or makes the cells more resistant to apoptotic stimuli.Apoptosis inhibitors include but are not limited to proteins, peptidesor protein- or peptide-like molecules, organic and inorganic molecules.Apoptosis inhibitors include compounds that act as metabolic inhibitors,inhibitors of nucleic acid degradation respectively nucleic acidpathways, enzyme inhibitors, in particular caspase inhibitors, calpaininhibitors and inhibitors of other enzymes involved in apoptoticprocesses. Respective apoptosis inhibitors are listed in Table 1.Preferably, the at least one apoptosis inhibitor that is used forstabilizing the cell-containing biological sample is selected from thegroup consisting of metabolic inhibitors, caspase inhibitors and calpaininhibitors. Suitable examples for each class are listed in Table 1 inthe respective category. Preferably, the apoptosis inhibitor iscell-permeable.

It is also within the scope of the present invention to use acombination of different apoptosis inhibitors, either from the same or adifferent class of apoptosis inhibitors, respectively to use acombination of different apoptosis inhibitors which inhibit apoptosiseither by the same or a different working mechanism.

In an advantageous embodiment of the present invention, the apoptosisinhibitor is a caspase inhibitor. Members of the caspase gene familyplay a significant role in apoptosis. The substrate preferences orspecificities of individual caspases have been exploited for thedevelopment of peptides that successfully compete caspase binding. It ispossible to generate reversible or irreversible inhibitors of caspaseactivation by coupling caspase-specific peptides to e.g. aldehyde,nitrile or ketone compounds. E.g. fluoromethyl ketone (FMK) derivatizedpeptides such as Z-VAD-FMK act as effective irreversible inhibitors withno added cytotoxic effects. Inhibitors synthesized with abenzyloxycarbonyl group (BOC) at the N-terminus and O-methyl side chainsexhibit enhanced cellular permeability. Further suitable caspaseinhibitors are synthesized with a phenoxy group at the C-terminus. Anexample is Q-VD-OPh which is a cell permeable, irreversiblebroad-spectrum caspase inhibitor that is even more effective inpreventing apoptosis than Z-VAD-FMK.

According to one embodiment, the caspase inhibitor is a pancaspaseinhibitor and thus is a broad spectrum caspase inhibitor. According toone embodiment, the caspase inhibitor comprises a modifiedcaspase-specific peptide. Preferably, said caspase-specific peptide ismodified by an aldehyde, nitrile or ketone compound. According to apreferred embodiment, the caspase specific peptide is modifiedpreferably at the carboxyl terminus with an O-Phenoxy or a fluoromethylketone (FMK) group. According to one embodiment, the caspase inhibitoris selected from the group consisting of Q-VD-OPh and Z-VAD(OMe)-FMK. Inone embodiment, Z-VAD(OMe)-FMK, a pancaspase inhibitor, is used, whichis a competitive irreversible peptide inhibitor and blocks caspase-1family and caspase-3 family enzymes. In a preferred embodiment,Q-VD-OPh, which is a broad spectrum inhibitor for caspases, is used.Q-VD-OPh is cell permeable and inhibits cell death by apoptosis.Q-VD-OPh is not toxic to cells even at extremely high concentrations andconsists of a carboxy terminal phenoxy group conjugated to the aminoacids valine and aspartate. It is equally effective in preventingapoptosis mediated by the three major apoptotic pathways, caspase-9 andcaspase-3, caspase-8 and caspase-10, and caspase-12 (Caserta et al,2003). Further caspase inhibitors are listed in Table 1. According toone embodiment, the caspase inhibitor that is used as apoptosisinhibitor for stabilizing the cell-containing sample is one which actsupon one or more caspases located downstream in the intracellular celldeath pathway of the cell, such as caspase-3. In one embodiment of thepresent invention the caspase inhibitor is an inhibitor for one or morecaspases selected from the group consisting of caspase-3, caspase-8,caspase-9, caspase-10 and caspase-12. It is also within the scope of thepresent invention to use a combination of caspase inhibitors.

The mixture that is obtained after contacting the biological sample withthe at least one apoptosis inhibitor may comprise the apoptosisinhibitor (or combination of apoptosis inhibitors) in a concentrationselected from the group of at least 0.01 μM, at least 0.05 μM, at least0.1 μM, at least 0.5 μM, at least 1 μM, at least 2.5 μM or at least 3.5μM. Of course, also higher concentrations can be used. Suitableconcentration ranges for the apoptosis inhibitor(s) when mixed with thecell-containing biological sample, include but are not limited to 0.01μM to 100 μM, 0.05 μM to 100 μM, 0.1 μM to 50 μM, 0.5 μM to 50 μM, 1 μMto 40 μM, more preferably 1 μM to 30 μM or 2.5 μM to 25 μM. The higherconcentrations were found to be more effective, however, goodstabilizing results were also achieved at lower concentrations. Hence,an efficient stabilization is also achieved at lower concentrations e.g.in a range selected from 0.1 μM to 10 μM, 0.5 μM to 7.5 μM or 1 μM to 5μM, in particular if the apoptosis inhibitor is used in combination witha hypertonic agent (see below). The above mentioned concentrations applyto the use of a single apoptosis inhibitor as well as to the use of acombination of caspase inhibitors. If a combination of caspaseinhibitors is used, the concentration of an individual apoptosisinhibitor that is used in said mixture of apoptosis inhibitors may alsolie below the above mentioned concentrations, if the overallconcentration of the combination of apoptosis inhibitors fulfils theabove mentioned features. Using a lower concentration that stillefficiently stabilizes the cells and/or reduce the degradation ofnucleic acids in present in the sample has the advantage that the costsfor stabilisation can be lowered. Lower concentrations can be used e.g.if the apoptosis inhibitor is used in combination with one or morestabilizers as described herein. The aforementioned concentrations arein particular suitable when using a caspase inhibitor, in particular amodified caspase specific peptide such as Q-VD-OPh and/or Z-VAD(OMe)-FMKas apoptosis inhibitor. The above mentioned concentrations are e.g. verysuitable for stabilizing whole blood, in particular 10 ml blood.Suitable concentration ranges for other apoptosis inhibitors and/or forother cell-containing biological samples can be determined by theskilled person using routine experiments, e.g. by testing the apoptosisinhibitors, respectively the different concentrations in the test assaysdescribed in the examples.

According to one embodiment, the apoptosis inhibitor will, in aneffective amount, decrease or reduce apoptosis in a cell-containingbiological sample by at least 25 percent, at least 30 percent, at least40 percent, at least 50 percent, preferably, by at least 75 percent,more preferably, by at least 85 percent as compared to a control samplewhich does not contain a respective apoptosis inhibitor.

According to a second sub-aspect of the first aspect of the presentinvention, at least one hypertonic agent is used for stabilizing thesample, wherein the used hypertonic agent stabilizes cells comprised inthe sample. As is shown by the provided examples, already the hypertonicagent alone is effective in stabilizing a cell-containing sample andsubstantially preserving the composition of the extracellular nucleicacid population comprised therein. The hypertonic agent induces cellshrinking by mild hypertonic effects (osmosis), thereby increasing thecell stability. Therefore, the cells are less prone to e.g. mechanicallyinduced cell damage. The sample can be contacted with the hypertonicagent, e.g. by adding the hypertonic agent to the sample or vice versa.The hypertonic agent present in the resulting mixture in particular issuitable for stabilizing cells contained in the sample, thereby reducingthe amount of intracellular nucleic acids, in particular genomic DNAthat is released from damaged cells. Thereby, the extracellular nucleicacid population is substantially preserved and the risk of contaminatingrespectively diluting the extracellular nucleic acids with intracellularnucleic acids, in particular genomic DNA, is reduced.

According to one embodiment, the hypertonic agent is sufficientlyosmotically active to induce cell shrinking (the cells release water),however, without damaging the cells i.e. without inducing or promotingcell lysis, respectively cell rupture. Hence, the hypertonic agentpreferably has a mild osmotic effect. Furthermore, it is desirous thatinteractions between the hypertonic agent and the sample arepredominantly limited to the cell stabilization effect basically inorder to avoid unwanted side effects. Thus, according to one embodiment,an uncharged hypertonic agent is used. Using an uncharged hypertonicagent has the advantage that even though the cells shrink respectivelyare stabilized due to the osmotic effect of the hypertonic agent,interactions between the hypertonic agent and other compounds comprisedin the sample are limited compared to the use of a charged hypertonicagent.

According to an advantageous embodiment, the hypertonic agent is ahydroxylated organic compound and accordingly, carries at least onehydroxyl group. According to one embodiment, the hydroxylated organiccompound comprises at least two hydroxyl groups. According to oneembodiment, the hydroxylated organic compound is a polyol. According toone embodiment, the polyol comprises 2 to 10 hydroxyl groups, preferably3 to 8 hydroxyl groups. The hydroxylated organic compound may comprise 2to 12 carbon atoms, preferably 3 to 8 and can be a cyclic or linearmolecule, branched or un-branched; it can be saturated or unsaturated;aromatic or non-aromatic. According to one embodiment, the hydroxylatedorganic compound is a hydroxy-carbonyl compound. A hydroxy-carbonylcompound is a compound possessing one or more hydroxy (OH) groups andone or more carbonyl groups. Hydroxylated organic compounds may includebut are not limited to hydroxylated ketone compounds and carbohydrates,or compounds derived therefrom. According to one embodiment, thehydroxylated organic compound is a polyalcohol, in particular a sugaralcohol. Hence, hydroxylated organic compounds include but are notlimited to carbohydrates such as glucose, raffinose, succrose, fructose,alpha-d-lactose monohydrate, inositol, maltitol, mannitol,dihydroxyacetone, alcohols such as glycerol, erythritol, mannitol,sorbitol, volemitol, or sugar alcohols. Suitable examples are alsolisted in the table below. It is also within the scope of the presentinvention to use combinations of respective hydroxylated organiccompounds.

Chemical Formula IUPAC Name Common Name Polyols, e.g. C₃H₅(OH)₃Propane-1,2,3-triol Glycerin C₄H₆(OH)₄ Butane-1,2,3,4-tetraol ErythritolC₅H₇(OH)₅ Pentane-1,2,3,4,5-pentol Xylitol, Arabitol, Ribitol C₆H₈(OH)₆Hexane-1,2,3,4,5,6-hexol Mannitol, Sorbitol, Dulcitol, Iditol C₇H₉(OH)₇Heptane-1,2,3,4,5,6,7- Volemitol heptol Alicyclic and sugar alcohols,e.g. C₆H₆(OH)₆ Cyclohexane-1,2,3,4,5,6- Inositol geksol C₁₂H₂₄O₁₁1-O-α-D-Glucopyranosyl-D- Isomalt mannitol C₁₂H₂₄O₁₁4-O-α-D-Glucopyranosyl-D- Maltitol glucitol C₁₂H₂₄O₁₁4-O-α-D-Galactopyranosyl- Lactitol D-glucitol

According to one embodiment, the polyols and sugar alcohols listed abovemay be replaced by alcohols with less hydroxyl groups (e.g.,hexane-1,2,3,4,5-pentol, pentane-1,2,3,4-tetraol). According to oneembodiment, the hydroxylated organic compound is no alcohol having 1 tocarbon atoms and carrying only one hydroxyl group. According to oneembodiment, alcohols with only one hydroxyl group are excluded ashydroxylated organic compound. The hydroxylated organic compound thatcan be used as stabilizer according to the present invention preferablyis water-soluble and non-toxic to the cells comprised in the biologicalsample to be stabilized. Preferably, the hydroxylated organic compounddoes not induce or support the lysis of the cells contained in thebiological sample and accordingly, preferably does not function as adetergent or as cell membrane dissolving agent. A suitable hydroxylatedorganic compound according to the present invention achieves astabilizing effect of the cell-containing sample by improving thepreservation of the composition of the extracellular nucleic acidpopulation as can be e.g. tested by the assays described in the examplesection.

Adding a hydroxylated organic compound to a cell-containing biologicalsample such as e.g. whole blood, increases the concentration of saidhydroxylated organic compound in the cell-free portion respectivelyfraction (e.g. the blood plasma) and thus forces blood cells to releasewater into the plasma as a result of an osmotic (hypertonic) effect.According to one embodiment, a hydroxylated organic compound is usedwhich is closely related to a product of the cell metabolism butpreferably can not be utilized by the cells.

According to a preferred embodiment, cells contained in the biologicalsample are essentially impermeable for the hypertonic agent that is usedfor stabilization. Thus, the hypertonic agent, which preferably is ahydroxylated organic compound as described in detail above, isessentially cell impermeable. Essentially cell impermeable in thisrespect in particular means that the concentration of the hypertonicagent, which preferably is a hydroxylated organic compound, issubstantially higher in the extracellular portion of the sample thaninside the cells contained in the biological sample that is stabilizedaccording to the teachings of the present invention. According to apreferred embodiment, the hypertonic agent, which preferably is ahydroxylated organic compound, is non-toxic, so that the cell viabilityis not compromised. This is preferred to avoid disturbing influences onthe cell metabolism.

According to one embodiment, the hypertonic agent is dihydroxyacetone(DHA). DHA is a carbohydrate and usually serves as tanning substance inself-tanning lotions. As is demonstrated by the examples, DHAsurprisingly has a remarkable stabilizing effect on cell-containingbiological samples, in particular whole blood samples and samplesderived from whole blood such as blood plasma or serum. DHA doesnaturally not occur in mammalian cells except for the phosphoric acidester of DHA, dihydroxyacetone-phosphat, an intermediate product ofglycolysis. Thus DHA is not expected to be actively transported or todiffuse into blood cells. According to one embodiment, the hypertonicagent is not dihydroxyaceton-phosphate.

The mixture that is obtained when contacting the cell-containingbiological sample with the at least one hypertonic agent may comprisethe hypertonic agent or mixture of hypertonic agents in a concentrationof at least 0.05M, preferably 0.1M, preferably at least 0.2M, morepreferred at least 0.25M. Of course, also higher concentrations can beused. Suitable concentration ranges for the hypertonic agent can beselected from 0.05M to 2M, 0.1M to 1.5M, 0.15M to 0.8M, 0.2M to 0.7M or0.1M to 0.6M. Respective concentrations are particularly suitable whenusing a hydroxylated organic compound, e.g. a carbohydrate such asdihydroxyacetone as hypertonic agent. The above mentioned concentrationsare e.g. very suitable for stabilizing whole blood, in particular 10 mlblood. Suitable concentration ranges for other hypertonic agents and/orother cell-containing biological samples can also be determined by theskilled person using routine experiments, e.g. by testing the hypertonicagents, respectively different concentrations thereof in the test assaysdescribed in the examples.

According to a third sub-aspect of the first aspect of the presentinvention, for stabilizing the extracellular nucleic acid population ina cell containing sample, at least one compound according to formula 1is used

wherein R1 is a hydrogen residue or an alkyl residue, preferably a C1-C5alkyl residue, more preferred a methyl residue, R2 and R3 are identicalor different hydrocarbon residues with a length of the carbon chain of1-20 atoms arranged in a linear or branched manner, and R4 is an oxygen,sulphur or selenium residue.

As is shown by the provided examples, a compound according to formula 1described above is effective in achieving a remarkable stabilizingeffect and in substantially preserving the composition of theextracellular nucleic acid population in the stabilized sample. Also amixture of one or more compounds according to formula 1 can be used forstabilization.

The hydrocarbon residues R2 and/or R3 can be selected independently ofone another from the group comprising alkyl, including short chain alkyland long-chain alkyl, alkenyl, alkoxy, long-chain alkoxy, cycloalkyl,aryl, haloalkyl, alkylsilyl, alkylsilyloxy, alkylene, alkenediyl,arylene, carboxylates and carbonyl. General groups, for instance alkyl,alkoxy, aryl etc. are claimed and described in the description and theclaims. Preferably, the following groups are used within the generallydescribed groups within the scope of the present invention:

-   (1) alkyl: preferably short chain alkyls, in particular linear and    branched C1-C5 alkyls or long-chain alkyls: linear and branched    C5-C20 alkyls;-   (2) alkenyl: preferably C2-C6 alkenyl;-   (3) cycloalkyl: preferably C3-C8 cycloalkyl;-   (4) alkoxy: preferably C1-C6 alkoxy;-   (5) long-chain alkoxy: preferably linear and branched C5-C20 alkoxy;-   (6) alkylenes: preferably a divalent linear or branched aliphatic,    cycloaliphatic or aromatic hydrocarbon residue with 2 to 18 carbon    atoms optionally containing heteroatoms, e.g. selected from the    group comprising: methylene; 1,1-ethylene; 1,1-propylidene;    1,2-propylene; 1,3-propylene; 2,2-propylidene; butan-2-ol-1,4-diyl;    propan-2-ol-1,3-diyl; 1,4-butylene; 1,4-pentylene; 1,6-hexylene;    1,7-heptylene; 1,8-octylene; 1,9-nonylene; 1,10-decylene;    1,11-undecylene; 1,12-docedylene; cyclohexane-1,1-diyl;    cyclohexane-1,2-diyl; cyclohexane-1,3-diyl; cyclohexane-1,4-diyl;    cyclopentane-1,1-diyl; cyclopentane-1,2-diyl; and    cyclopentane-1,3-diyl;-   (7) alkenediyl: preferably selected from the group comprising:    1,2-propenediyl; 1,2-butenediyl; 2,3-butenediyl; 1,2-pentenediyl;    2,3-pentenediyl; 1,2-hexenediyl; 2,3-hexenediyl; and 3,4-hexenediyl;-   (8) alkynediyl: is equal to —C≡C—;-   (9) aryl: preferably selected from aromatics with a molecular weight    below 300 Da;-   (10)arylenes: preferably selected from the group comprising:    1,2-phenylene; 1,3-phenylene; 1,4-phenylene; 1,2-naphtthalenylene;    1,3-naphtthalenylene; 1,4-naphtthalenylene; 2,3-naphtthalenylene;    1-hydroxy-2,3-phenylene; 1-hydroxy-2,4-phenylene;    1-hydroxy-2,5-phenylene; 1-hydroxy-2,6-phenylene;-   (11)carboxylate: preferably the group —C(O)OR, where R is selected    from: hydrogen; C1-C6 alkyl; phenyl; C1-C6 alkyl-C₆H₅; Li; Na; K;    Cs; Mg; Ca;-   (12)carbonyl: preferably the group —C(O)R, where R is selected from:    hydrogen; C1-C6 alkyl; phenyl; C1-C6 alkyl-C6H5 and amine (resulting    in an amide) selected from the group: —NR′2, where each R′ is    selected independently from: hydrogen; C1-C6 alkyl; C1-C6 alkyl-C6H5    and phenyl, where, if both Rs represent C1-C6 alkyl they can form an    NC3 to NC5 heterocyclic ring with alkyl substituents of the ring    forming the other alkyl chain;-   (13)alkylsilyl: preferably the group —SiR1R2R3, where R1, R2 and R3    are selected independently of one another from: hydrogen; alkyl;    long-chain alkyl; phenyl; cycloalkyl; haloalkyl; alkoxy; long-chain    alkoxy;-   (14)alkylsilyloxy: preferably the group —O—SiR1R2R3, where R1, R2    and R3 are selected independently of one another from: hydrogen;    alkyl; long-chain alkyl; phenyl; cycloalkyl; haloalkyl; alkoxy;    long-chain alkoxy.

The chain length n of R2 and/or R3 can in particular have the values 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20.Preferably R2 and R3 have a length of the carbon chain of 1-10. In thiscase the chain length n can in particular have the values 1, 2, 3, 4, 5,6, 7, 8, 9, and 10. Preferably, R2 and R3 have a length of the carbonchain of 1-5 and in this case the chain length can in particular havethe values 1, 2, 3, 4 and 5. Particularly preferred is a chain length of1 or 2 for R2 and R3.

The chain length n of R1 preferably has the value 1, 2, 3, 4 or 5.Particularly preferred is a chain length of 1 or 2 for R1.

R4 preferably is oxygen.

According to a preferred embodiment, the compound according to formula 1is a N,N-dialkyl-carboxylic acid amide. Preferred R1, R2, R3 and R4groups are described above. According to one embodiment, the compound isselected from the group consisting of N,N-dimethylacetamide;N,N-diethylacetamide; N,N-dimethylformamide and N,N-diethylformamide.Also suitable are N,N-dialkylpropanamides such asN,N-dimethylpropanamide as is shown in the examples. Preferably, thesubstance according to formula 1 is N,N-dimethlylacetamide (DMAA). Thestructural formulae of the preferred compounds are as follows:

Also suitable are the respective thio analogues, which comprise sulphurinstead of oxygen as R4.

The mixture that is obtained when contacting the cell-containingbiological sample with a compound according to formula 1 or a mixture ofrespective compounds may comprise said compound or mixture of compoundsin a final concentration of at least 0.1%, at least 0.5%, at least0.75%, at least 1%, at least 1.25% or at least 1.5%. A suitableconcentration range includes but is not limited to 0.1% up to 50%.Preferred concentration ranges can be selected from the group consistingof 0.1% to 30%, 0.1% to 20%, 0.1% to 15%, 0.1% to 10%, 0.1% to 7.5%,0.1% to 5%, 1% to 30%, 1% to 20%, 1% to 15%, 1% to 10%, 1% to 7.5%, 1%to 5%; 1.25% to 30%, 1.25% to 20%, 1.25% to 15%, 1.25% to 10%, 1.25% to7.5%, 1.25% to 5%; 1.5% to 30%, 1.5% to 20%, 1.5% to 15%, 1.5% to 10%,1.5% to 7.5% and 1.5% to 5%. Respective concentrations are particularlysuitable when using a N,N-dialkyl-carboxylic acid amide, e.g.N,N-dimethylacetamide, N,N-diethylacetamide, N,N-diethylformamide orN,N-diemethylformamide or N,N-dimethylpropanamide as stabilizing agent.The above mentioned concentrations are e.g. very suitable forstabilizing whole blood or blood products such as plasma. Suitableconcentration ranges for other compounds according to formula 1 and/orother cell-containing biological samples can also be determined by theskilled person using routine experiments, e.g. by testing the compound,respectively different concentrations thereof in the test assaysdescribed in the examples.

Preferably, the compound according to formula 1 is used in combinationwith a chelating agent for stabilizing the cell containing sample. Inparticular, a chelating agent can be used as anticoagulant whenstabilizing a blood sample or a sample derived from blood such as e.g.plasma or serum. Suitable chelating agents and concentration ranges areprovided below.

According to a preferred fourth sub-aspect, a method suitable forstabilizing a cell-containing sample, preferably a blood sample isprovided, wherein said method comprises contacting the sample with

-   -   a) at least one apoptosis inhibitor, and    -   b) at least one hypertonic agent, which stabilizes the cells        comprised in the sample.

Thus, according to this preferred embodiment, the apoptosis inhibitorand the hypertonic agent, which both alone are already effective instabilizing a cell-containing sample (see above and examples), are usedin combination. Thereby, the stabilization effect can be increasedand/or the concentration of the individual components (the apoptosisinhibitor and/or the hypertonic agent) may also be reduced while stillefficiently preserving the extracellular nucleic acid population in thesample, and in particular avoiding, respectively reducing thecontamination by intracellular nucleic acids in particular fragmentedgenomic DNA that is released from damaged or decaying cells contained inthe sample. As is shown in the examples, using a respective combinationis particularly effective in stabilizing a cell-containing sample, evenvery complex samples such as a whole blood sample. It is also within thescope of the present invention to use a mixture of different apoptosisinhibitors in combination with different hypertonic agents. Suitable andpreferred embodiments of the apoptosis inhibitor and the hypertonicagent as well as suitable and preferred concentrations of the respectiveagents suitable for achieving an efficient stabilization of the sampleare described in detail above in conjunction with the embodiments,wherein either an apoptosis inhibitor or a hypertonic agent is used tostabilize the cell-containing biological sample. It is referred to theabove disclosure which also applies to the embodiment, wherein anapoptosis inhibitor is used in combination with a hypertonic agent.Preferably, at least one caspase inhibitor, preferably a modifiedcaspase specific peptide, preferably modified at the C-terminus with anO-phenoxy group such as Q-VD-OPh, is used in combination with at leastone hydroxylated organic compound, e.g. a carbohydrate, such asdihydroxyacetone or a polyol, as hypertonic agent. As is demonstrated bythe examples, a respective combination is remarkably effective instabilizing a cell-containing biological sample, in particular a wholeblood sample, at room temperature for more than 3 days and even for 6days.

According to one embodiment, a combination of stabilizing agents is usedwhich comprises at least one apoptosis inhibitor, at least onehypertonic agent and/or at least one compound according to formula 1 asdefined above. Examples of respective combinations include (1) acombination of at least one apoptosis inhibitor and at least onecompound according to formula 1 as defined above, (2) a combination ofat least one hypertonic agent and at least one compound according toformula 1 as defined above or (3) a combination of all three stabilizingagents, i.e. at least one apoptosis inhibitor, at least one hypertonicagent and at least one compound according to formula 1 as defined above.A respective combination may also comprise additional additives thatenhance the stabilizing effect such as e.g. anticoagulants and chelatingagents. According to one embodiment, the combination of stabilizingagents comprises a caspase inhibitor and an anticoagulant, preferably achelating agent such as EDTA. Respective combinations can be accordingto a fifth sub-aspect advantageously used in a method suitable forstabilizing an extracellular nucleic acid population comprised in acell-containing sample according to the first aspect of the presentinvention. The stabilizing effect observed with combinations ofstabilizing agents is stronger than the effect observed for any of theindividual stabilizing agents when used alone and/or allows to use lowerconcentrations, thereby making combinatorial use of stabilizing agentsan attractive option. Suitable and preferred embodiments of theapoptosis inhibitor, the hypertonic agent and the compound according toformula 1 defines above as well as suitable and preferred concentrationsof the respective agents suitable for achieving an efficientstabilization of the sample are described in detail above in conjunctionwith the embodiments, wherein either an apoptosis inhibitor, ahypertonic agent or a compound according to formula 1 is used tostabilize the cell-containing biological sample.

As discussed in the background of the invention, extracellular nucleicacids are usually not present “naked” in the sample but are e.g.stabilized to a certain extent by being released protected in complexesor by being contained in vesicles and the like. This has the effect thatextracellular nucleic acids are already to a certain extent stabilizedby nature and thus, are usually not degraded rapidly by nucleases incell-containing samples such as whole blood, plasma or serum. Thus, whenintending to stabilize extracellular nucleic acids that are comprised ina biological sample, one of the primary problems is the dilution,respectively the contamination of the extracellular nucleic acidpopulation by intracellular nucleic acids, in particular fragmentedgenomic DNA, that originates from damaged or dying cells that arecontained in the sample. This also poses a problem when processingcell-depleted samples such as plasma or serum (which are sometimes alsodescribes as being “cell-free” even though they may comprise minoramounts of cells). The stabilization technology according to the presentinvention is of particular advantage in this respect because it not onlysubstantially preserves the extracellular nucleic acids present in thesample and e.g. inhibits degradation of the comprised extracellularnucleic acids (preferably at least by 60%, at least by 70%, at least by75%, at least by 80%, at least by 85%, at least by 90% or mostpreferably at least by 95% over the stabilization period compared to anunstabilized sample or an EDTA stabilized sample) but furthermore,efficiently reduces the release of genomic DNA from cells contained inthe sample and/or reduces the fragmentation of respective genomic DNA.According to one embodiment, using the apoptosis inhibitor, thehypertonic agent and/or the compound according to formula 1 forstabilizing the cell-containing sample according to the teachings of thepresent invention has the effect that the increase of DNA that resultsfrom a release of DNA from cells contained in the sample is reducedcompared to a non-stabilized sample. According to one embodiment, saidrelease of genomic DNA is reduced by at least 3-fold, at least 4-fold,at least 5-fold, at least 6-fold, at least 7-fold, at least 10-fold, atleast 12-fold, at least 15-fold, at least 17-fold or at least 20-foldover the stabilization period compared to the non-stabilized sample or acorresponding sample that is stabilized with EDTA (in particular in caseof a blood sample or a sample derived from blood such as plasma orserum). According to one embodiment, said release of genomic DNA isreduced by at least 60%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90% or at least 95% over the stabilization periodcompared to the non-stabilized sample or a corresponding sample that isstabilized with EDTA (in particular in case of a blood sample or asample derived from blood such as plasma or serum). The release of DNAcan be determined e.g. by quantifying the ribosomal 18S DNA as isdescribed herein in the example section. E.g. standard EDTA stabilizedblood samples show a 40-fold increase of DNA determined e.g. at day 6 ofstorage at room temperature in a respective assay (see FIG. 2b ). Thestabilization achievable with the teachings of the present inventionremarkably reduces this release of DNA even down to e.g. a maximum of4-fold. Thus, the extracellular nucleic acid population contained in thesample is considerably stabilized compared to samples stabilized instandard EDTA tubes. Thus, according to one embodiment, thestabilization effect that is achieved with the apoptosis inhibitor, thehypertonic agent and/or the compound according to formula 1 as taught bythe present invention results in that the release of DNA from cellscontained in the sample is at least reduced to a maximum of 10-fold,preferably 7-fold, more preferably 5-fold and most preferably is atleast reduced to a maximum of 4-fold, as is e.g. determinable in the 18SDNA assay described in the examples. As is shown by the examples, aneffective stabilization of the extracellular nucleic acid population isachievable for a period of at least up to 6 days. During a shorterstorage of the samples, e.g. up to three days, the DNA release can bereduced at least to a maximum of two-fold as e.g. determinable in the18S DNA assay described in the examples. Thus, the DNA release can bereduced to 2 fold or less up to three days of storage when using thestabilizing methods according to the present invention. This is aremarkable improvement in the stabilization of the extracellular nucleicacid population compared to the prior art methods. This significantlyenhances the accuracy of any subsequent tests. In certain cases, forexample if the sample material has to be transported for long distancesor stored for longer periods e.g. at room temperature (as can be e.g.the case in certain countries), the process according to the inventionmakes it possible for the first time for these tests to be carried outafter such a period of time. However, of course, the samples may also befurther processed earlier, if desired. It is not necessary to make useof the full achievable stabilization period. The stabilization that isachieved with the present invention reduces variations in theextracellular nucleic acid population that may result from a differenthandling/processing of the samples (e.g. storage conditions and periods)after they were collected. This greatly improves the standardization ofhandling and molecular analysis.

Further additives may be used in addition to the apoptosis inhibitor,the hypertonic agent and/or the compound according to formula 1 asdefined above in order to further stabilize the cell-containing sample.The selection of suitable additives that may also contribute to thestabilization effect may also depend on the type of cell-containingsample to be stabilized. E.g. when processing whole blood ascell-containing biological sample, it is advantageous and also common toinclude an anticoagulant e.g. selected from the group consisting ofheparin, ethylenediamine tetraacetic acid, citrate, oxalate, and anycombination thereof. In an advantageous embodiment, the anticoagulant isa chelating agent. A chelating agent is an organic compound that iscapable of forming coordinate bonds with metals through two or moreatoms of the organic compound. Chelating agents according to the presentinvention include, but are not limited to diethylenetriaminepentaaceticacid (DTPA), ethylenedinitrilotetraacetic acid (EDTA), ethylene glycoltetraacetic acid (EGTA) and N,N-bis(carboxymethyl)glycine (NTA).According to a preferred embodiment, EDTA is used. As used herein, theterm “EDTA” indicates inter alia the EDTA portion of an EDTA compoundsuch as, for example, K₂EDTA, K₃EDTA or Na₂EDTA. Using a chelating agentsuch as EDTA also has the advantageous effect that nucleases such asDNases are inhibited, thereby e.g. preventing a degradation ofextracellular DNA by DNases. Furthermore, it was found by the inventorsthat EDTA used/added in higher concentrations is capable of reducing therelease of intracellular nucleic acids, in particular genomic DNA fromthe cells thereby supporting the stabilizing effect that is achieved bythe apoptosis inhibitor, the hypertonic agent and/or the at least onecompound according to formula 1. However, EDTA alone is not capable ofefficiently inhibiting the fragmentation of e.g. genomic DNA that isreleased from the cells contained in the sample. Thus, EDTA does notachieve a sufficient stabilization effect. But used in combination withthe teachings of the present invention, in particular in combinationwith the apoptosis inhibitor, in particular the caspase inhibitor, itcan further improve the stabilization for the above discussed reasons.Furthermore, it also appears to increase the chemical stability of RNA.According to one embodiment, the concentration of the chelating agent,preferably EDTA, in the biological sample that is mixed with one or moreof the stabilizing compounds described above is in the range selectedfrom the group consisting of 0.05 mM to 100 mM, 0.05 mM to 50 mM, 0.1 mMto 30 mM, 1 mM to 20 mM and 2 mM to 15 mM after the contacting step.Respective concentrations are particularly effective when stabilisingblood, plasma and/or serum samples, in particular 10 ml blood samples.

Additional additives can also be used in order to further support thestabilization of the cell-containing sample, respectively support thepreservation of the extracellular nucleic acid population. Examples ofrespective additives include but are not limited to nuclease inhibitors,in particular RNase and DNase inhibiting compounds. Examples of RNaseinhibitors include but are not limited to anti-nuclease antibodies orribonucleoside-vanadyl-complexes. When choosing a respective furtheradditive, care should be taken not to compromise and/or counteract thestabilizing effect of the apoptosis inhibitor, the hypertonic agentand/or the compound according to formula 1. Thus, no additives should beused in concentrations that result in or support the lysis and/ordegradation of the cells contained in the biological sample and/or whichsupport the degradation of the nucleic acids contained in the cell-freefraction of the biological sample.

In an advantageous embodiment of the present invention, thecell-containing biological sample, which preferably is a blood sample ora sample derived from blood such as plasma or serum, is contacted with:

-   a) at least one caspase inhibitor as an apoptosis inhibitor,    preferably with Q-VD-OPh, preferably in a concentration range of 1    μM to 30 μM;-   b) optionally at least one hydroxylated organic compound such as    dihydroxyacetone as hypertonic agent, preferably in a concentration    range of 0.1M to 0.6M; and-   c) optionally at least one compound according to formula 1 defined    above (preferred embodiments and concentrations are described above)    and/or-   d) a further additive, preferably a chelating agent preferably in a    concentration range of 4 mM to 50 mM, preferably 4 mM to 20 mM, most    preferably EDTA.

The components of the stabilizing composition can be comprised,respectively dissolved in a buffer, e.g. a biological buffer such asMOPS, TRIS, PBS and the like.

The apoptosis inhibitor, the hypertonic agent and/or the compoundaccording to formula 1 as defined above as well as the optionallypresent further additives can be e.g. present in a device, preferably acontainer, for collecting the sample or can be added to a respectivecollection device immediately prior to collection of the biologicalsample; or can be added to the collection device immediately after thesample was collected therein. It is also within the scope of the presentinvention to add the stabilizing agent(s) and optionally, the furtheradditive(s) separately to the cell containing biological sample.However, for the ease of handling, it is preferred that the one or morestabilizing agents and optionally the further additives are provided inone composition. Furthermore, in an advantageous embodiment, theapoptosis inhibitor, the hypertonic agent and/or the compound accordingto formula 1 as described above and optionally the further additive(s)are present in the collection device prior to adding the sample. Thisensures that the cell-containing biological sample is immediatelystabilized upon contact with the stabilizing agent(s). The stabilisationagent(s) are present in the container in an amount effective to providethe stabilisation of the amount of cell containing sample to becollected, respectively comprised in said container. As described, thesample can be mixed with the stabilization agent(s) directly afterand/or during collection of the sample thereby providing a stabilizedsample.

Preferably, the sample is mixed with the stabilization agent(s) directlyafter and/or during the collection of the sample. Therefore, preferably,the stabilization agent(s) and additives described above are provided inform of a stabilizing composition. Preferably, said stabilizingcomposition is provided in liquid form. It can be e.g. pre-filled in thesample collection device so that the sample is immediately stabilizedduring collection. According to one embodiment, the stabilizingcomposition is contacted with the cell-containing sample in a volumetricratio selected from 10:1 to 1:20, 5:1 to 1:15, 1:1 to 1:10 and 1:2 to1:5. It is a particular advantage of the teachings of the presentinvention that stabilization of a large sample volume can be achievedwith a small volume of the stabilizing composition. Therefore,preferably, the ratio of stabilizing composition to sample lies in arange from 1:2 to 1:7, more preferred 1:3 to 1:5.

The term “cell-containing sample” as used herein, in particular refersto a sample which comprises at least one cell. The cell-containingsample may comprise at least two, at least 10, at least 50, at least100, at least 250, at least 500, at least 1000, at least 1500, at least2000 or at least 5000 cells. Furthermore, also cell-containing samplescomprising considerably more cells are encompassed by said term and canbe stabilized with the teachings according to the present invention.However, the term “cell-containing sample” also refers to and thusencompasses cell-depleted samples, including cell-depleted samples thatare commonly referred to as “cell-free” such as e.g. blood plasma asrespective samples often include residual cells. At least, it can oftennot be fully excluded that even so-called “cell-free” samples such asblood plasma comprise residual amounts of cells which accordingly, posea risk that the extracellular nucleic acid population becomescontaminated with intracellular nucleic acids released from saidresidual cells. Therefore, respective cell-depleted and “cell-free”samples are according to one embodiment also encompassed by the term“cell-containing sample”. Thus, the “cell-containing sample” maycomprise large amounts of cells, as is the case e.g. with whole blood,but may also only comprise merely minor amounts of cells. Hence, theterm “cell containing sample” also encompasses samples that may only besuspected of or pose a risk of containing cells. As discussed above,also with respect to biological samples which only comprise minor,respectively residual amounts of cells such as e.g. blood plasma (bloodplasma contains—depending on the preparation method—usually smallresidual amounts of cells, even though it is commonly referred to asbeing cell-free), the method according to the present invention hasconsiderable advantages as these residual cells may also result in aundesired contamination of the comprised extracellular nucleic acids.Using the stabilizing technology of the present invention also ensuresthat respective samples which only comprise residual amounts of cells orare merely suspected of or pose a risk of residual amounts of cells, areefficiently stabilized as is also described in detail above. Using thestabilizing method according to the present invention has the advantagethat irrespective of the composition of the sample and the number ofcells contained therein, the extracellular nucleic acid populationcontained therein is substantially preserved, respectively stabilized,thereby allowing for standardizing the subsequent isolation and/oranalysis of the contained extracellular nucleic acids.

According to one embodiment, the cell-containing biological sample isselected from the group consisting of whole blood, samples derived fromblood, plasma, serum, sputum, lachrymal fluid, lymphatic fluid, urine,sweat, liquor, cerebrospinal fluid, ascites, milk, stool, bronchiallavage, saliva, amniotic fluid, nasal secretions, vaginal secretions,semen/seminal fluid, wound secretions, and cell culture supernatants andsupernatants obtained from other swab samples. According to oneembodiment, the cell-containing biological sample is a body fluid, abody secretion or body excretion, preferably a body fluid, mostpreferably whole blood, plasma or serum. The cell-containing biologicalsample comprises extracellular nucleic acids. According to anotherembodiment, the cell-containing biological sample is a non-fluid samplederived from a human or animal, such as e.g. stool, tissue or a biopsysample. Other examples of cell-containing biological samples that can bestabilized with the method according to the present invention includebut are not limited to biological samples cell suspensions, cellcultures, supernatant of cell cultures and the like, which compriseextracellular nucleic acids.

As is described above and as is demonstrated by the examples, using themethods of the present invention allows for stabilizing thecell-containing sample without refrigeration or freezing for a prolongedperiod of time period. Thus, the samples can be kept at room temperatureor even at elevated temperatures e.g. up to 30° C. or up to 40° C.According to one embodiment, a stabilization effect is achieved for atleast two days, preferably at least three days; more preferred at leastone day to six days, most preferred for at least one day to at leastseven days at room temperature. As is shown in the examples, the samplesthat were stabilized according to the method of the present inventionwere not substantially compromised when stored for 3 days at roomtemperature. Even during longer storages for up to 6 or even 7 days atroom temperature the extracellular nucleic acid population wassubstantially more stabilized compared to non-stabilized samples or e.g.compared to samples that were stabilized using standard method such asan EDTA treatment. Even though the stabilization effect may decreaseover time, it is still sufficient to preserve the composition of theextracellular nucleic acid population to allow the analysis and/orfurther processing. Thus, samples that were stabilized according to themethods of the present invention were still suitable for isolating andoptionally analysing the extracellular nucleic acids contained thereineven after longer storage at room temperature. Thus, as the samples werenot compromised in particular when using the preferred combination ofstabilisation agents, even longer storage/shipping times areconceivable. However, usually, longer periods are not necessary, as theregular storage and e.g. shipping time to the laboratory, wherein thenucleic acid isolation and optionally analysis is performed, usuallydoes not exceed 6 or 7 days, but usually is even completed after two orthree days. As is shown in the examples, the stabilisation efficiency isparticularly good during this time period. However, the extraordinarylong stabilisation times and stabilisation efficiencies that areachievable with the method according to the present invention providesan important safety factor.

The methods and also the subsequently described compositions accordingto the present invention allow the stabilization also of large volumesof biological samples with small volumes of added substances because theadditives that are used according to the teachings of the presentinvention are highly active. This is an important advantage because thesize/volume of the sample poses considerable restrains on the subsequentisolation procedure in particular when intending to use automatedprocesses for isolating the extracellular nucleic acids contained in thesamples. Furthermore, one has to consider that extracellular nucleicacids are often only comprised in small amounts in the contained sample.Thus, processing larger volumes of a cell-containing sample such as e.g.a blood sample has the advantage that more circulating nucleic acids canbe isolated from the sample and thus are available for a subsequentanalysis.

The stabilization of the biological sample may either be followeddirectly by techniques for analysing nucleic acids, or the nucleic acidsmay be purified from the sample. Hence, the sample that was stabilizedaccording to the method of the present invention can be analysed in anucleic acid analytic and/or detection method and or may be furtherprocessed. E.g. extracellular nucleic acid can be isolated from thestabilized sample and can then be analysed in a nucleic acid analyticand/or detection method or may be further processed.

Furthermore, according to a second aspect, a method for isolatingextracellular nucleic acids from a cell-containing biological sample isprovided, wherein said method comprises the steps of:

-   -   a) stabilizing the extracellular nucleic acid population        comprised in a cell-containing sample according to the method        defined in the first aspect of the present invention;    -   b) isolating extracellular nucleic acids.

As discussed above, the stabilization according to the present inventionhas the effect that the extracellular nucleic acid population containedin the sample is substantially preserved in the state it had shown atthe time the biological sample was obtained, respectively drawn. Inparticular, the usually observed high increase in nucleic acids thatresults from intracellular nucleic acids, in particular genomic DNA,more specifically fragmented genomic DNA, released from damaged or dyingcells is efficiently reduced as is demonstrated in the examples.Therefore, the extracellular nucleic acids obtained from a respectivelystabilized sample comprise fewer contaminations with intracellularnucleic acids originating from degraded or dying cells comprised in thesample and in particular comprise less amounts of fragmented genomic DNAcompared to non-stabilized samples. Furthermore, the uniquestabilization step allows to increase the amount of recoverableextracellular nucleic acids. The stabilization method according to thepresent invention can be performed without the crosslinking of thesample. This is an important advantage over the use of cross-linkingagents such as formaldehyde or formaldehyde releasers, as these reagentsmight reduce the recoverable amount of extracellular nucleic acids dueto cross-linking. Thus, the method according to the present inventionimproves the diagnostic and prognostic capability of the extracellularnucleic acids. Furthermore, said stabilization allows the sample to bestored and/or handled, e.g. transported,—even at room temperature—for aprolonged period of time prior to separating the cells contained in thesample and/or prior to isolating the extracellular nucleic acidscomprised therein in step b). With respect to the details of thestabilization, it is referred to the above disclosure which also applieshere.

According to one embodiment, the cell-containing biological sample suchas e.g. a whole blood sample is stabilized in step a) as is described indetail above using at least one apoptosis inhibitor, at least onehypertonic agent and/or at least one compound according to formula 1 asdescribed above, preferably using at least two of these stabilizingagents and optionally, further additives. Suitable and preferredembodiments were described above. Particularly preferred is the use of acaspaseinhibitor in combination with an anticoagulant, preferably achelating agent as described above, for stabilizing whole blood samples.

If the sample comprises large amounts of cells as is e.g. the case withwhole blood, the cells are separated from the remaining sample in orderto obtain a cell-free, respectively cell-reduced or cell-depletedfraction of the sample which comprises the extracellular nucleic acids.Thus, according to one embodiment, cells are removed from thecell-containing sample between step a) and step b). This intermediatestep is only optional and e.g. may be obsolete if samples are processedwhich merely comprise minor amounts of residual cells such as e.g.plasma or serum. However, in order improve the results it is preferredthat also respective remaining cells (or potentially remaining cells)are removed as they might contaminate the extracellular nucleic acidpopulation during isolation. Depending on the sample type, cells,including residual cells, can be separated and removed e.g. bycentrifugation, preferably high speed centrifugation, or by using meansother than centrifugation, such as e.g. filtration, sedimentation orbinding to surfaces on (optionally magnetic) particles if acentrifugation step is to be avoided. Respective cell removal steps canalso be easily included into an automated sample preparation protocol.Respectively removed cells may also be processed further. The cells cane.g. be stored and/or biomolecules such as e.g. nucleic acids orproteins can be isolated from the removed cells.

Furthermore, it is also within the scope of the present invention toinclude further intermediate steps to work up the sample.

Extracellular nucleic acids are then isolated in step b), e.g. from thecell-free, respectively cell-depleted fraction, e.g. from supernatants,plasma and/or serum. For isolating extracellular nucleic acids, anyknown nucleic acid isolation method can be used that is suitable forisolating nucleic acids from the respective sample, respectively thecell-depleted sample. Examples for respective purification methodsinclude but are not limited to extraction, solid-phase extraction,silica-based purification, magnetic particle-based purification,phenol-chloroform extraction, chromatography, anion-exchangechromatography (using anion-exchange surfaces), electrophoresis,filtration, precipitation, chromatin immunoprecipitation andcombinations thereof. It is also within the scope of the presentinvention to specifically isolate specific target extracellular nucleicacids, e.g. by using appropriate probes that enable a sequence specificbinding and are coupled to a solid support. Also any other nucleic acidisolating technique known by the skilled person can be used. Accordingto one embodiment, the nucleic acids are isolated using a chaotropicagent and/or alcohol. Preferably, the nucleic acids are isolated bybinding them to a solid phase, preferably a solid phase comprisingsilica or anion exchange functional groups. Suitable methods and kitsare also commercially available such as the QIAamp® Circulating NucleicAcid Kit (QIAGEN), the Chemagic Circulating NA Kit (Chemagen), theNucleoSpin Plasma XS Kit (Macherey-Nagel), the Plasma/Serum CirculatingDNA Purification Kit (Norgen Biotek), the Plasma/Serum Circulating RNAPurification Kit (Norgen Biotek), the High Pure Viral Nucleic Acid LargeVolume Kit (Roche) and other commercially available kits suitable forextracting and purifying circulating nucleic acids.

According to one embodiment, all nucleic acids that are comprised in thesample that is obtained after step a) or optionally obtained after thecells have been removed in the intermediate step are isolated, e.g. areisolated from the cell-free, respectively cell-depleted fraction. E.g.total nucleic acids can be isolated from plasma or serum and theextracellular nucleic acids will be comprised as a portion in theseextracted nucleic acids. If the cells are efficiently removed, the totalnucleic acids isolated will predominantly comprise or even consist ofextracellular nucleic acids. It is also within the scope of the presentinvention to isolate at least predominantly a specific target nucleicacid. A target nucleic acid can be e.g. a certain type of nucleic acid,e.g. RNA or DNA, including mRNA, microRNA, other non-coding nucleicacids, epigenetically modified nucleic acids, and other nucleic acids.It is also within the scope of the present invention to e.g. digest thenon-target nucleic acid using nucleases after isolation. The term targetnucleic acid also refers to a specific kind of nucleic acid, e.g. aspecific extracellular nucleic acid that is known to be a certaindisease marker. As discussed above, the isolation of extracellularnucleic acids may also comprise the specific isolation of a respectivetarget nucleic acid e.g. by using appropriate capture probes. The term atarget nucleic acid also refers to a nucleic acid having a certainlength, e.g. a nucleic acid having a length of 2000 nt or less, 1000 ntor less or 500 nt or less. Isolating respective smaller target nucleicacids can be advantageous because it is known that extracellular nucleicacids usually have a smaller size of less than 2000 nt, usually lessthan 1000 nt and often even less than 500 nt. The sizes, respectivelysize ranges indicated herein refer to the chain length. I.e. in case ofDNA it refers to bp. Focusing the isolation, respectively purification,on respective small nucleic acids can increase the portion ofextracellular nucleic acids obtained in the isolated nucleic acids. Thestabilization methods according to the present invention allow, inparticular due to the inhibition of fragmentation of genomic,intracellular DNA, for a more efficient separation of such highmolecular weight genomic DNA from the fragmented extracellular nucleicacid population, e.g., during the nucleic acid extraction procedure. Asthe substantial size difference between genomic and circulating nucleicacids is essentially preserved using the stabilization technologyaccording to the present invention, genomic DNA can be removed e.g. bysize-selective recovery of DNA more efficiently than without therespective stabilization. Suitable methods to achieve a respectiveselective isolation of the extracellular nucleic acid population e.g. bydepleting the high molecular weight genomic DNA are well-known in theprior art and thus, need no further description here. E.g. it would besufficient to use a size-selection method that depletes a sample of anynucleic acid larger than 1,000-10,000 nucleotides or base pairs. As thesize difference between genomic (usually larger than >10,000 bp) andextracellular nucleic acids (usually <1000 bp) in a stabilized sampleaccording to the present invention is usually relatively large due tothe efficient stabilization (the difference can e.g. lie in a range of1000-10,000 bp), known methods for selectively isolating extracellularnucleic acid from a biological sample could be applied. This alsoprovides further opportunities in order to reduce the amount ofintracellular nucleic acids in the isolated extracellular nucleic acidpopulation. For example, the removal of genomic DNA during the nucleicacid extraction protocol could also supplement or even replace aseparate high g-force centrifugation of a plasma sample before startingthe nucleic acid extraction in order to remove residual cells. GenomicDNA that is released from said residual cells is prevented from becomingmassively degraded due to the stabilization according to the presentinvention, and accordingly, can be removed by size-selective isolationprotocols. This option is of particular advantage, as many clinicallaboratories do not have a centrifuge capable of performing such a highg-force centrifugation or other means for removing in particular traceamounts of residual cells.

The isolated nucleic acids can then be analysed and/or further processedin a step c) using suitable assay and/or analytical methods. E.g. theycan be identified, modified, contacted with at least one enzyme,amplified, reverse transcribed, cloned, sequenced, contacted with aprobe, be detected (their presence or absence) and/or be quantified.Respective methods are well-known in the prior art and are commonlyapplied in the medical, diagnostic and/or prognostic field in order toanalyse extracellular nucleic acids (see also the detailed descriptionin the background of the present invention). Thus, after extracellularnucleic acids were isolated, optionally as part of total nucleic acid,total RNA and/or total DNA (see above), they can be analysed to identifythe presence, absence or severity of a disease state including but notbeing limited to a multitude of neoplastic diseases, in particularpremalignancies and malignancies such as different forms of cancers.E.g. the isolated extracellular nucleic acids can be analysed in orderto detect diagnostic and/or prognostic markers (e.g., fetal- ortumor-derived extracellular nucleic acids) in many fields ofapplication, including but not limited to non-invasive prenatal genetictesting respectively screening, disease screening, pathogen screening,oncology, cancer screening, early stage cancer screening, cancer therapymonitoring, genetic testing (genotyping), infectious disease testing,injury diagnostics, trauma diagnostics, transplantation medicine or manyother diseases and, hence, are of diagnostic and/or prognosticrelevance. According to one embodiment, the isolated extracellularnucleic acids are analyzed to identify and/or characterize a disease ora fetal characteristic. Thus, as discussed above, the isolation methoddescribed herein may further comprise a step c) of nucleic acid analysisand/or processing. Therefore, according to one embodiment, the isolatedextracellular nucleic acids are analysed in step c) to identify, detect,screen for, monitor or exclude a disease and/or at least one fetalcharacteristic. The analysis/further processing of the nucleic acids canbe performed using any nucleic acid analysis/processing methodincluding, but not limited to amplification technologies, polymerasechain reaction (PCR), isothermal amplification, reverse transcriptionpolymerase chain reaction (RT-PCR), quantitative real time polymerasechain reaction (Q-PCR), digital PCR, gel electrophoresis, capillaryelectrophoresis, mass spectrometry, fluorescence detection, ultravioletspectrometry, hybridization assays, DNA or RNA sequencing, restrictionanalysis, reverse transcription, NASBA, allele specific polymerase chainreaction, polymerase cycling assembly (PCA), asymmetric polymerase chainreaction, linear after the exponential polymerase chain reaction(LATE-PCR), helicase-dependent amplification (HDA), hot-start polymerasechain reaction, intersequence-specific polymerase chain reaction (ISSR),inverse polymerase chain reaction, ligation mediated polymerase chainreaction, methylation specific polymerase chain reaction (MSP),multiplex polymerase chain reaction, nested polymerase chain reaction,solid phase polymerase chain reaction, or any combination thereof.Respective technologies are well-known to the skilled person and thus,do not need further description here.

According to one embodiment, either or both of the isolating oranalyzing steps b) and c) occurs at least one day up to 7 days after thesample has been collected, respectively stabilized according to theteachings of the present invention. Suitable time periods for which thesample, in particular a blood sample, respectively the extracellularnucleic acid population contained therein can be stabilized using themethod according to the present invention are also described above andalso apply here. According to one embodiment, the isolation step isperformed at least one day, at least 2 days, at least 3 days, at least 4days, at least 5 days or at least 6 days after the sample was collectedand stabilized according to the method according to the presentinvention. According to one embodiment, either or both of the isolatingor analyzing steps occur without freezing the sample and/or without theuse of formaldehyde for preserving the cell-containing biologicalsample. The biological sample is stabilized after the contact with theapoptosis inhibitor, the hypertonic agent and/or the compound accordingto formula 1 as defined above, preferably in combination with a furtheradditive such as an anticoagulant like EDTA. An anticoagulant ispreferably used when stabilizing blood or a sample derived from blood.The respectively stabilized samples can be handled, e.g. stored and/orshipped at room temperature.

Furthermore, according to a third aspect of the present invention acomposition suitable for stabilizing the extracellular nucleic acidpopulation in a biological sample is provided, comprising:

-   -   a) at least one apoptosis inhibitor, preferably a caspase        inhibitor, and/or    -   b) at least one hypertonic agent which stabilizes cells        comprised in the sample, preferably dihydroxyacetone; and/or    -   c) at least one compound according to formula 1 as defined        above; and    -   d) optionally at least one anticoagulant, preferably a chelating        agent.

As discussed above, a respective stabilizing composition is particularlyeffective in stabilizing a cell-containing biological sample, inparticular whole blood, plasma and/or serum by stabilizing the comprisedcells and the comprised extracellular nucleic acids therebysubstantially preserving, respectively stabilizing the extracellularnucleic acid population. A respective stabilizing composition allows thestorage and/or handling, e.g. shipping, of the sample, which preferablyis whole blood, at room temperature for at least two, preferably atleast three days without substantially compromising the quality of thesample, respectively the extracellular nucleic acid population containedtherein. Of course, it is not mandatory to make use of the full possiblestabilization period; the samples may also be processed earlier ifdesired. Contacting the biological sample with the stabilizingcomposition allows the sample to be stored, and or handled, e.g.shipped, even at room temperature prior to isolating and optionallyanalysing and/or processing the contained circulating nucleic acids.Thus, the time between the collection or stabilization of the sample andthe nucleic acid extraction can vary without substantially affecting thepopulation, respectively the composition of the extracellular nucleicacid population contained therein. In particular, dilutions,respectively contaminations with intracellular nucleic acids, inparticular fragmented genomic DNA, are reduced. Preferably, thestabilization composition is contacted with the sample immediately afteror during collection of the sample. Preferably, when stabilizing a bloodsample, the composition comprises at least one caspase inhibitor and atleast one anticoagulant, preferably a chelating agent as describedabove. It may also comprise further stabilizing agents as describedherein.

Suitable and preferred embodiments of the apoptosis inhibitor, thehypertonic agent and/or the compound according to formula 1 as well assuitable and preferred concentrations of the respective compounds aredescribed in detail above in conjunction with the stabilization method.It is referred to the above disclosure which also applies with respectto the stabilization composition. Preferably, at least one caspaseinhibitor, preferably a modified caspase specific peptide, preferablymodified at the C-terminus with an O-phenoxy group such as Q-VD-OPh, isused in combination with at least one hypertonic agent, preferably ahydroxylated organic compound such as dihydroxyacetone. Other suitablehydroxylated organic compounds are also described above, it is referredto the respective disclosure. As is demonstrated by the examples, arespective combination is remarkably effective in stabilizing acell-containing biological sample, in particular a blood sample.

Preferably, the at least one compound according to formula 1 is aN,N-dialkyl-carboxylic acid amide. Preferred R1, R2, R3 and R4 groupsare described above. According to one embodiment, the compound isselected from the group consisting of N,N-dimethylacetamide;N,N-diethylacetamide; N,N-dimethylformamide, N,N-diethylformamide andN,N-dimethylpropanamid. Said compound can also be used in combinationwith an apoptosis inhibitor, preferably a caspase inhibitor (preferredembodiments are described above, it is referred to the above disclosure)and/or a hypertonic agent, preferably a hydrxycarbon compound (preferredembodiments are described above, it is referred to the abovedisclosure).

Furthermore, it is preferred that the stabilization compositioncomprises further additives, e.g. an anticoagulant such as a chelatingagent in particular if the composition is used for stabilizing wholeblood, plasma or serum.

According to one embodiment, the stabilizing composition consistsessentially of the mentioned stabilizers and optional additives andoptionally, buffering agents. The stabilizing composition stabilizes thesample and thus, does not promote the lysis and/or disruption of thecells contained in the sample. The stabilizing composition may reducethe damage of the cells comprised in the sample as can be e.g.determined by the assay methods described in the example section.

The composition may be provided in a solid form. This is e.g. a suitableoption if the biological sample to be stabilized contains liquid todissolve the solid (such as for example cell-containing body fluids,cells in medium, urine) or if liquid, e.g. water is added thereto todissolve the solid. The advantage of using a solid stabilizingcomposition is that solids are usually chemically more stable. However,also a liquid composition may be used. Liquid compositions often havethe advantage that the mixture with the sample to be stabilised can bequickly achieved, thereby basically providing an immediate stabilisingeffect as soon as the sample comes into contact with the liquidstabilizing composition. Preferably, stabilising agent(s) present in theliquid stabilizing composition remain stable in solution and require nopre-treatment-such as for example the dissolving of precipitates oflimited solubility-by the user because pre-treatments of this kind posea risk of variations in the stabilising efficiency.

Also provided is a mixture comprising the stabilizing compositionaccording to the present invention mixed with a biological sample.Suitable and preferred examples of biological samples as well assuitable concentrations of the stabilizing agent(s) when mixed with thebiological sample are described above in conjunction with thestabilizing method. It is referred to the above disclosure which alsoapplies here. Preferably, the stabilizing composition is pre-filled in asample collection device so that the sample is immediately stabilizedduring collection. According to one embodiment, the stabilizingcomposition is contacted with the biological sample in a volumetricratio selected from 10:1 to 1:20, 5:1 to 1:15, 1:1 to 1:10 and 1:2 to1:5. It is a particular advantage of the stabilizing composition of thepresent invention that stabilization of a large sample volume can beachieved with a small volume of the stabilizing composition. Therefore,preferably, the ratio of stabilizing composition to sample lies in arange from 1:2 to 1:7, more preferred 1:3 to 1:5.

The stabilizing composition according to the third aspect of the presentinvention can be used to stabilize the extracellular nucleic acidpopulation comprised in a cell-containing sample. Furthermore, thestabilizing composition according to the third aspect of the presentinvention may also be used for stabilizing cells contained in a sample.As described above, the stabilizing composition inter alia reduces therelease of genomic DNA from cells that results from decaying cells.Thus, a respective use is also an advantageous and provided by theteachings according to the present invention.

Also provided is a method of manufacturing a composition according tothe third aspect of the present invention is provided, wherein thecomponents of the composition are mixed, preferably in an aqueoussolution.

The composition of the present invention may also be incorporated into asample collection device, in particular blood collection assembly,thereby providing for a new and useful version of such a device. Suchdevices typically include a container having an open and a closed end.The container is preferably a blood collection tube. The container typealso depends on the sample to be collected, other suitable formats aredescribed below.

Furthermore, the present invention provides a container for collecting acell-containing biological sample, preferably a blood sample, whereinthe container comprises a stabilizing composition according to thepresent invention. Providing a respective container, e.g. a samplecollection tube, which comprises the stabilizing composition accordingto the present invention, has the advantage that the sample is quicklystabilized when the sample is collected in the respective container.Details with respect to the stabilizing composition were describedabove, it is referred to the above disclosure which also applies here.

According to one embodiment, a collection container for receiving andcollecting a biological sample is provided wherein the containercomprises:

-   a) at least one apoptosis inhibitor such that when the sample is    collected, the concentration of the apoptosis inhibitor or    combination of two or more apoptosis inhibitors in the resulting    mixture is selected from at least 0.01 μM, at least 0.05 μM, at    least 0.1 μM, at least 0.5 μM, at least 1 μM, at least 2.5 μM or at    least 3.5 μM and preferably is present in a concentration range    selected from 0.01 μM to 100 μM, 0.05 μM to 100 μM, 0.1 μM to 50 μM,    1 μM to 40 μM, 1.0 μM to 30 μM or 2.5 μM to 25 μM and/or-   b) at least one hypertonic agent such that when the sample is    collected, the concentration of the hypertonic agent or combination    of two or more apoptosis inhibitors in the resulting mixture is at    least 0.05M, at least 0.1M, preferably at least 0.25M, and    preferably is present in a concentration range from 0.05M to 2M,    0.1M to 1.5M, 0.15M to 0.8M, 0.2M to 0.7M or 0.1M to 0.6M; and/or-   c) at least one compound according to formula 1 as defined above,    such that when the sample is collected the compound according to    formula 1 is comprised in a concentration of at least 0.1%, at least    0.5%, at least 0.75%, at least 1%, at least 1.25% or at least 1.5%    or wherein said compound is comprised in a concentration range    selected from 0.1% up to 50%. 0.1 to 30%, 1% to 20%, 1% to 10%, 1%    to 7.5% and 1% to 5%; and/or-   d) optionally at least one further additive, preferably an    anticoagulant such as a chelating agent, preferably EDTA if the    container is for collecting blood or a blood product. Suitable    concentrations are described above and preferably lie in the range    of 4 mM to 50 mM, more preferred 4 mM to 20 mM.

The pre-filled components a), b), c) and/or d) can be provided in aliquid or in a dry form. For stabilizing whole blood it is preferred touse at least components a) and d). Preferably, the stabilizingcomponents are provided as a stabilizing composition. A dry form is e.g.a suitable option if the biological sample to be stabilized containsliquid to dissolve the solid (such as for example cell-containing bodyfluids, cells in medium, urine) or if liquid, e.g. water is addedthereto to dissolve the solid. The advantage of using a solidstabilizing composition is that solids are usually chemically morestable than liquids. According to one embodiment, the inner wall of thecontainer is treated/covered with a stabilizing composition according tothe present invention. Said composition can be applied to the innerwalls using e.g. a spray-dry-method. Liquid removal techniques can beperformed on the stabilising composition in order to obtain asubstantially solid state protective composition. Liquid removalconditions may be such that they result in removal of at least about 50%by weight, at least about 75% by weight, or at least about 85% by weightof the original amount of the dispensed liquid stabilising composition.Liquid removal conditions may be such that they result in removal ofsufficient liquid so that the resulting composition is in the form of afilm, gel or other substantially solid or highly viscous layer. Forexample it may result in a substantially immobile coating (preferably acoating that can be re-dissolved or otherwise dispersed upon contactwith the cell-containing sample which preferably is a blood productsample). It is possible that lyophilization or other techniques may beemployed for realizing a substantially solid form of the protectiveagent (e.g., in the form of one or more pellet). Thus, liquid removalconditions may be such that they result in a material that upon contactwith the sample under consideration (e.g., a whole blood sample) theprotective agent will disperse in the sample, and substantially preservecomponents (e.g., extracellular nucleic acids) in the sample. Liquidremoval conditions may be such that they result in a remainingcomposition that is substantially free of crystallinity, has a viscositythat is sufficiently high that the remaining composition issubstantially immobile at ambient temperature; or both.

However, also a liquid composition may be used. Liquid compositionsoften have the advantage that the mixture with the sample to bestabilised can be quickly achieved, thereby basically providing animmediate stabilising effect as soon as the sample comes into contactwith the liquid stabilizing composition. Preferably, the stabilisingagent(s) present in the liquid stabilizing composition remain stable insolution and require no pre-treatment—such as for example the dissolvingof precipitates of limited solubility—by the user because pre-treatmentsof this kind pose a risk of variations in the stabilising efficiency.

The stabilizing composition is comprised in the container in an amounteffective to provide the stabilisation of the amount of sample to becollected in said container. According to one embodiment, the liquidstabilizing composition is contacted with the biological sample in avolumetric ratio selected from 10:1 to 1:20, 5:1 to 1:15, 1:1 to 1:10and 1:2 to 1:5. It is a particular advantage of the stabilizingcomposition of the present invention that stabilization of a largesample volume can be achieved with a small volume of the stabilizingcomposition. Therefore, preferably, the ratio of stabilizing compositionto sample lies in a range from 1:2 to 1:7, more preferred 1:3 to 1:5.

According to one embodiment, the container is evacuated. The evacuationis preferably effective for drawing a specific volume of a fluid sampleinto the interior. Thereby, it is ensured that the correct amount ofsample is contacted with the pre-filled amount of the stabilizingcomposition comprised in the container, and accordingly, that thestabilization is efficient. According to one embodiment, the containercomprises a tube having an open end sealed by a septum. E.g. thecontainer is pre-filled with a defined amount of the stabilizingcomposition either in solid or liquid form and is provided with adefined vacuum and sealed with a septum. The septum is constructed suchthat it is compatible with the standard sampling accessories (e.g.cannula, etc.). When contacted with e.g. the canula, a sample amountthat is predetermined by the vacuum is collected in the container. Arespective embodiment is in particular advantageous for collectingblood. A suitable container is e.g. disclosed in U.S. Pat. No.6,776,959.

The container according to the present invention can be made of glass,plastic or other suitable materials. Plastic materials can be oxygenimpermeable materials or may contain an oxygen impermeable layer.Alternatively, the container can be made of water- and air-permeableplastic material. The container according to the present inventionpreferably is made of a transparent material. Examples of suitabletransparent thermoplastic materials include polycarbonates,polyethylene, polypropylene and polyethyleneterephthalate. The containermay have a suitable dimension selected according to the required volumeof the biological sample being collected. As described above,preferably, the container is evacuated to an internal pressure belowatmospheric pressure. Such an embodiment is particularly suitable forcollecting body fluids such as whole blood. The pressure is preferablyselected to draw a predetermined volume of a biological sample into thecontainer. In addition to such vacuum tubes also non-vacuum tubes,mechanical separator tubes or gel-barrier tubes can be used as samplecontainers, in particular for the collection of blood samples. Examplesof suitable containers and capping devices are disclosed in U.S. Pat.No. 5,860,397 and US 2004/0043505. As container for collecting thecell-containing sample also further collection devices, for example asyringe, a urine collection device or other collection devices can beused. The type of the container may also depend on the sample type to becollected and suitable containers are also available to the skilledperson.

In an advantageous embodiment the container respectively the device isfilled or is pre-filled with at least one apoptosis inhibitor,preferably a caspase inhibitor, at least one hypertonic agent,preferably at least one hydroxylated organic compound as described indetail above, e.g. dihydroxyaceton and optionally a further additivesuch as an anticoagulant, preferably a chelating agent, more preferredEDTA. The mixture of at least one hypertonic agent, which preferably isa hydroxylated organic compound, e.g. a carbohydrate such asdihydroxyacetone and at least one caspase inhibitor, preferablyQ-VD-OPH, unexpectedly stabilizes extracellular nucleic acids in wholeblood, plasma or serum and prevents the release of cellular nucleicacids in particular from white blood cells that are contained in suchsamples. Hence, the extracellular nucleic acid population is preservedin the state it had shown at the time of blood draw. Beneficial resultsare also obtained when the container respectively the device is filledor is pre-filled with at least one compound according to formula 1 asdefined above as stabilizing agent. Preferably, an anticoagulant isencompassed in addition to the compound according to formula 1. Theanticoagulant is preferably a chelating agent such as EDTA. Furthermore,the stabilizing composition comprised in the container may also comprisean apoptosis inhibitor, preferably a caspase inhibitor and/or at leastone hypertonic agent, preferably at least one hydroxylated organiccompound as described in detail above, e.g. dihydroxyaceton andoptionally further additives. According to one embodiment, thestabilizing composition comprised in the container comprises a caspaseinhibitor and an anticoagulant.

According to one embodiment, the container has an open top, a bottom,and a sidewall extending therebetween defining a chamber, wherein thestabilization composition according to the present invention iscomprised in the chamber. It may be comprised therein in liquid or solidform. According to one embodiment the container is a tube, the bottom isa closed bottom, the container further comprises a closure in the opentop, and the chamber is at a reduced pressure. The advantages of areduced pressure in the chamber were described above. Preferably, theclosure is capable of being pierced with a needle or cannula, and thereduced pressure is selected to draw a specified volume of a liquidsample into the chamber.

According to one embodiment, the chamber is at a reduced pressureselected to draw a specified volume of a liquid sample into the chamber,and the stabilizing composition is a liquid and is disposed in thechamber such that the volumetric ratio of the stabilising composition tothe specified volume of the cell-containing sample is selected from 10:1to 1:20, 5:1 to 1:15, 1:1 to 1:10 and 1:2 to 1:5. The associatedadvantages were described above.

Preferably, the container is for drawing blood from a patient.

According to a fifth aspect, a method is provided comprising the step ofcollecting a sample from a patient directly into a chamber of acontainer according to the fourth aspect of the present invention.Details with respect to the container and the sample were describedabove. It is referred to the respective disclosure. According to oneembodiment, a blood sample is collected, preferably it is withdrawn fromthe patient.

The methods and compositions disclosed herein allow for the efficientpreservation and isolation of extracellular nucleic acids while reducingpossible mixing with nucleic acids, in particular fragmented genomicDNA, which originates from cells comprised in the biological sample andwhich may enter a biological sample due to cell damage, respectivelycell lysis. The methods according to the present invention, as well asthe compositions and the disclosed devices (e.g. the collectioncontainers) reduce the degradation of extracellular nucleic acids andalso reduce cell lysis and/or release of genomic nucleic acids, inparticular fragmented genomic DNA, so that the extracellular nucleicacids contained in the sample do not become contaminated withintracellular nucleic acids, respectively a respective contamination isreduced by the teachings according to the present invention. Asdiscussed above, an intermixing of extracellular nucleic acids andcellular nucleic acids, in particular fragmented genomic DNA, may reducethe accuracy of any measurement of the amount of extracellular nucleicacids in a biological sample. As discussed above, an important advantageof the present invention is the possibility for essentially simultaneousstabilizing of both the cells contained in the sample (in particularwhite blood cells in case of whole blood, plasma or serum) and theextracellular nucleic acids. This helps to prevent cellular nucleicacids such as genomic DNA from being released into the cell-free portionof the sample, and further diluting the comprised extracellular nucleicacids (and associated biomarkers) of interest, while also maintainingthe structural integrity of the extracellular nucleic acids. Asdiscussed herein, contacting the cell-containing biological sample suchas whole blood or plasma with the stabilising agent(s) allows the sampleto be stored for a period of time prior to isolating the extracellularnucleic acids. More preferably, the cell-containing biological sample,e.g. blood or plasma, may be drawn at one location (e.g., a health carefacility), contacted with the stabilising agent(s), and latertransported to a different remote location (e.g., a laboratory) for thenucleic acid isolation and testing process.

Furthermore, the stabilization reagents, as disclosed in herein, providean advantage over known state-of-the-art stabilization reagents whichinvolve the use of cross-linking reagents, such as formaldehyde,formaldehyde releasers and the like, as the stabilization of samplesaccording to the present invention does not involve the use to suchcrosslinking reagents. Crosslinking reagents cause inter- orintra-molecular covalent bonds between nucleic acid molecules or betweennucleic acids and proteins. This effect can lead to a reduced recoveryof such stabilized and partially crosslinked nucleic acids after apurification or extraction from a complex biological sample. As, forexample, the concentration of circulating nucleic acids in a whole bloodsamples is already relatively low, any measure which further reduces theyield of such nucleic acids should be avoided. This may be of particularimportance when detecting and analyzing very rare nucleic acid moleculesderived from malignant tumors or from a developing fetus in the firsttrimester of pregnancy. Therefore, according to one embodiment, noformaldehyde releaser is comprised in the stabilizing composition,respectively is not additionally used for stabilization. According toone embodiment, the apoptosis inhibitor that is used in the methodsand/or compositions according to the present invention is not selectedfrom the group consisting of aurintricarboxylic acid,phenylmethylsulfonyl fluoride (PMSF), leupeptin and Na-Tosyl-Lyschloromethyl ketone hydrochloride (TLCK). According to one embodiment,the apoptosis inhibitor is not selected from said group in particular ifthe apoptosis inhibitor is not used in combination with a hypertonicagent as additional stabilizer.

This invention is not limited by the exemplary methods and materialsdisclosed herein, and any methods and materials similar or equivalent tothose described herein can be used in the practice or testing ofembodiments of this invention. Numeric ranges are inclusive of thenumbers defining the range. The headings provided herein are notlimitations of the various aspects or embodiments of this inventionwhich can be read by reference to the specification as a whole.

The term “solution” as used herein in particular refers to a liquidcomposition, preferably an aqueous composition. It may be a homogenousmixture of only one phase but it is also within the scope of the presentinvention that a solution comprises solid additives such as e.g.precipitates.

The sizes, respectively size ranges indicated herein with reference tonucleotides nt, refer to the chain length and thus are used in order todescribe the length of single-stranded as well as double-strandedmolecules. In double-stranded molecules said nucleotides are paired.

According to one embodiment, subject matter described herein ascomprising certain steps in the case of methods or as comprising certainingredients in the case of compositions, solutions and/or buffers refersto subject matter consisting of the respective steps or ingredients. Itis preferred to select and combine preferred embodiments describedherein and the specific subject-matter arising from a respectivecombination of preferred embodiments also belongs to the presentdisclosure.

TABLE 1 Overview of apoptosis inhibitors Apoptosis inhibitorDescription 1. Metabolic inhibitors AlCA-Riboside, Acadesine, Offersprotection against cell death induced by glucose deprivation AlCAr,5-Aminoimidazole-4- carboxamide-1-β-riboside, Z- Riboside ApoptosisInhibitor II, diarylurea prevents the active ~700-kDa apoptosome complexformation compound Bax Channel Blocker, (±)-1- A cell-permeabledibromocarbazolo-piperazinyl derivative that(3,6-Dibromocarbazol-9-yl)-3- displays anti-apoptotic properties.Effectively blocks Bid-induced piperazin-1-yl-propan-2-ol, biscyctochrome c release from HeLa cell mitochondria (~80% TFA, iMAC1inhibition at 5 μM) by inhibiting Bax channel-forming activity (IC50 =520 nM in a liposome channel assay). Bax-Inhibiting Peptide, V5 Acell-permeable pentapeptide based on the Ku70-Bax inhibiting Peptidesequence: domain that offers cytoprotection. Functions as effectively asthe H-Val-Pro-Met-Leu-Lys-OH Caspase Inhibitor VI (Z-VAD-FMK; Cat. No.219007) for Bax- (SEQ ID NO: 1) mediated apoptosis (~50-200 μM). Alsoeffectively blocks caspase- independent necrotic cell death. Shown to beKu70 competitive, interact with Bax, prevent its conformational changeand mitochondrial translocation. Displays extended stability in culturemedium (~3 days). Negative control peptide is also available Bcl-xLBH44-23, Human, Cell- A cell-permeable peptide that prevents apoptoticcell death by Permeable directly binding to the voltage-dependent anionchannel (VDAC) and blocking its activity. Leads to the inhibition ofcytochrome c release and loss of mitochondrial membrane potential (ΔΨm).Contains the conserved N-terminal homology domain (BH4) of Bcl- xL(amino acids 4-23) that has been shown to be essential for inhibitingVDAC activity in liposomes and in isolated mitochondria. The BH4 domainis linked to a carrier peptide, a 10-amino acid HIV-TAT48-57 sequencewith a β-alanine residue as a spacer for maximum flexibility. Followingits uptake, it is mainly localized to the mitochondria Bongkrekic Acid,Triammonium Acts as a ligand of the adenine nucleotide translocator. Apotent Salt inhibitor of mitochondrial megachannel (permeabilitytransition pore). Significantly reduces signs of apoptosis induced bynitric oxide. Prevents the apoptotic breakdown of the innermitochondrial transmembrane potential (ΔΨm), as well as a number ofother phenomena linked to apoptosis Daunorubicin, Hydrochloride Potentcell-permeable anticancer agent whose potential target site may bemitochondrial cytochrome c oxidase. Has been shown to inhibit RNA andDNA synthesis. Inhibits eukaryotic topoisomerases I and II. Induces DNAsingle-strand breaks. Also induces apoptosis in HeLa S3 tumor cells.According to one embodiment, said compound is not used as stabilizeraccording to the present invention. Humanin, Human, Synthetic A24-residue anti-apoptotic peptide that, when expressed intracellularly,offers protection against neuronal apoptosis induced by presenilin andAPP (amyloid precursor protein) mutants associated with familialAlzheimer's disease (AD). Shown to reduce cytochrome c release in vitroby directly binding to Bax (Bcl-2-associated X protein; Kd ~2 nM) andpreventing its association with isolated mitochondriaPhorbol-12-myristate-13-acetate Most commonly-used phorbol ester.Extremely potent mouse skin tumor promoter. Activates protein kinase Cin vivo and in vitro, even at nM concentrations. Promotes the expressionof inducible NOS in cultured hepatocytes. Activates Ca2+-ATPase andpotentiates forskolin-induced cAMP formation. Inhibits apoptosis inducedby the Fas antigen, but induces apoptosis in HL-60 promyelocyticleukemia cells. Its binding is reversible Pifithrin-α A cell-permeablechemical inhibitor of p53. Reversibly inhibits p53- dependenttransactivation of p53-responsive genes and reversibly blocksp53-mediated apoptosis. Inhibits p53-dependent growth arrest of humandiploid fibroblasts in response to DNA damage but has no effect onp53-deficient fibroblasts. Protects normal tissues from the deleteriousside effects of chemotherapy. Has been reported to protect neuronsagainst β-amyloid peptide and glutamate-induced apoptosis Pifithrin-μ Acell-permeable sulfonamide that blocks p53 interaction with Bcl- xL andBcl-2 proteins and selectively inhibits p53 translocation tomitochondria without affecting the transactivation function of p53.Effectively protects against γ radiation-induced cell death in vitro andanimal lethality in vivo. Because Pifithrin-μ targets only themitochondrial branch of the p53 pathway without affecting the importanttranscriptional functions of p53, it is superior to Pifithrin-α (Cat.No. 506132) in in vivo studies. Shown to selectively interact withinducible HSP70 and disrupt its functions Pifithrin-α, Cyclic- Acell-permeable and very stable analog of Pifithrin-α (Cat. No. 506132),with similar biological function, but with reduced cytotoxicity. Achemical inhibitor of p53. Reversibly inhibits p53- dependenttransactivation of p53-responsive genes; also reversibly blocksp53-mediated apoptosis. Acts as a P-gp modulator by changing relativesubstrate specificity of the transporter. This compound has beenreported to be a potent STAT6 transcriptional inhibitor Pifithrin-α,p-Nitro A cell-permeable p53 inhibitor that serves as the prodrug formof Pifithrin-α, p-Nitro, Cyclic (Cat. No. 506154). Although its in vitroefficacy (ED50 = 0.3 μM in protecting etoposide-induced cortical neurondeath) is similar to that of Pifithrin-α (Cat. No. 506132), it is100-fold more potent than Pifithrin-α when adminstered in rats in vivodue to its long-lasting, steady conversion to the corresponding cyclicform of active compound in biological systems (t½ = 8 h in neuronculture medium at 37° C.). Pifithrin-α, p-Nitro, Cyclic A cell-permeablep53 inhibitor that exhibits 10-fold higher potency (ED50 = 30 nM inprotecting etoposide-induced cortical neuron death) and 50% longerhalf-life (t½ = 6 h in neuron culture medium at 37° C.) than Pifithrin-α(Cat. No. 506132). However, despite its in vitro efficacy, thisinhibitor is not effective when adminstered in rats in vivo. For in vivoapplications, please consider Pifithrin-α, p-Nitro (Cat. No. 506152).STAT3 Inhibitor Peptide A Stat3-SH2 domain binding phosphopeptide thatacts as a Peptide sequence: selective inhibitor of Stat3 (signaltransducers and activators of Ac-Pro-Tyr(PO3H2)-Leu-Lys- transcription3) signaling with a DB50 of 235 μM (concentration of Thr-Lys-OH peptideat which DNA-binding activity is inhibited by 50%). (SEQ ID NO: 2)Significantly lowers the DNA-binding activity of Stat3 by forming aninactive Stat3:peptide complex and reduces the levels of activeStat3:Stat3 dimers that can bind DNA. Displays greater affinity forStat3, and to a lesser extent Stat1, over Stat5. Supplied as atrifluoroacetate salt. STAT3 Inhibitor Peptide, Cell- A cell-permeableanalog of the Stat3-SH2 domain-binding Permeable phosphopeptide (Cat.No. 573095) that contains a C-terminal mts Peptide sequence: (membranetranslocating sequence) and acts as a highly selective,Ac-Pro-Tyr(PO3H2)-Leu-Lys- potent blocker of Stat3 activation. Alsosuppresses constitutive Thr-Lys-OH Stat-3 dependent Src transformationwith no effect on Stat-3 (SEQ ID NO: 2) independent Ras transformation.The unphosphorylated inactive control peptide is also available underCat. No. 573105. Supplied as a trifluoroacetate salt. CAY10500,6,7-dimethyl-3- Tumor necrosis factor α (TNFα) inhibitor that preventsbinding to {[methyl-[1-(3-trifluoromethyl- the TNF Receptor 1 (TNFR1).6Binds to the biologically active phenyl)-1H-indol-3-ylmethyl]- TNFαtrimer and promotes accelerated displacement of a singleamino}-ethyl)-amino]-methyl}- subunit to rapidly inactivate thecytokine. In a cell based assay, chromen-4-one compound inhibitedTNFα-mediated stimulation of IKB degradation. Gambogic amide A selectiveagonist for TrkA which mimics the actions of NGF. This compoundpossesses robust neurotrophic actvity, while it prevents neuronal celldeath 1. Maslinic Acid A pentacyclic triterpene with antioxidant andanti-inflammatory properties. Shown to block the generation of nitricoxide, and inhibits the secretion of IL-6 and TNF-α induced bylipopolysaccharides Naringin hydrate A citrus bioflavonoid found toinhibit cytochrome P450 monooxygenase activity in mouse liver. Itprevents toxin-induced cytoskeletal disruption and apoptotic liver celldeath. Necrostatin-1 An inhibitor of necroptosis, a non-apoptotic celldeath pathway. Does not affect Fas/TNFR-triggered apoptosis. Accordingto one embodiment, said compound is not used as stabilizer according tothe present invention. NSC348884 hydrate, N1,N2- This product is anucleolar phosphoprotein that displays severalbis((3-imino-6-methyl-3H-indol- biological activities in ribosomebiogenesis, cell proliferation, 2-yl)methyl)-N1,N2-bis((6-cytoplasmic/nuclear shuttle transportation, nucleic acid binding,methyl-1H-benzo[d]imidazol-2- ribonucleic cleavage, centrosomeduplication and molecular yl)methyl)ethane-1,2-diamine chaperoning, andis found in higher levels in tumor cells. hydrate Overexpression hasbeen shown to lead to inhibition of apoptosis. NSC34884 upregulates p53.Orsellinic acid Benzoic acid. Blocks PAF-mediated neuronal apoptosis.Shows free radical scavenging activity. tetramethyl A syntheticderivative of NDGA and a non-selective lipoxygenase NordihydroguaiareticAcid inhibitor. It inhibits Sp1 transcription factor binding at the HIVlong terminal repeat promoter and at the α-ICP4 promoter (a geneessential for HSV replication). GW 4869, 3,3′-(1,4- A cell-permeable,symmetrical dihydroimidazolo-amide compound phenylene)bis[N-[4-(4,5-that acts as a potent, specific, non-competitive inhibitor of N-dihydro-1H-imidazol-2- SMase (neutral sphingomyelinase) [IC50 = ~1 μM,rat brain; Km yl)phenyl]-hydrochloride-2- for sphingomyelin ~13 μM].Does not inhibit human A-SMase (acid propenamide sphingomyelinase) evenat 150 μM. Weakly inhibits the activities of bovine protein phosphatase2A and mammalian lyso-PAF PLC, while no inhibition is observed forbacterial phosphatidylcholine- specific PLC. Reported to offer completeprotection against TNF-α or diamine-induced cell death in MCF7 breastcancer cells at 20 μM. Does not modify the intracellular glutathionelevels or interfere with TNF-α or diamine-mediated signaling effects. SP600125, 1,9- SP600125 is a JNK inhibitor (IC50 = 40 nM for JNK-1 andJNK-2 Pyrazoloanthrone, and 90 nM for JNK-3). This agent exhibitsgreater than 300-fold Anthrapyrazolone selectivity for JNK againstrelated MAP kinases ERK1 and p38-2, and the serine threonine kinase PKA.[1] SP600125 is a reversible ATP-competitive inhibitor. In cells,SP600125 dose dependently inhibited the phosphorylation of c-Jun, theexpression of inflammatory genes COX-2, IL-2, IFN-γ, TNF-α, andprevented the activation and differentiation of primary human CD4 cellcultures Mdivi-1, 3-(2,4-Dichloro-5- Mdivi-1 is a selective inhibitor ofmitochondrial division in yeast and methoxyphenyl)-2,3-dihydro-2-mammalian cells which acts via inhibiting the mitochondrial divisionthioxo-4(1H)-quinazolinone, 3- dynamin. In cells, Mdivi-1 inhibitsapoptosis by inhibiting (2,4-Dichloro-5-methoxyphenyl)- mitochondrialouter membrane permeabilization. Mdivi-1 causes2-sulfanyl-4(3H)-quinazolinone the rapid (<5 min) reversible anddose-dependent formation of net- like mitochondria in wild-type cellswith an IC50 = ~10 μM. In yeast, time-lapse fluorescence microscopyrevealed no detectable mitochondrial division after treatment withMdivi-1 Minocycline•hydrochloride Tetracycline derivative withantimicrobial activity. Inhibitor of angiogenesis, apoptosis andpoly(ADP-ribose) polymerase-1 (PARP-1). Anti-inflammatory andneuroprotective Ro 08-2750 (C13H10N4O3) Inhibitor of NGF-inducedapoptosis. RKTS-33 (C7H8O4) selective inhibition of Fas ligand-dependentpathway alone 2. Nucleic acids 3,4-Dichloroisocoumarin Inhibitor ofserine proteases −> granzyme B and blocks apoptotic internucleosomal DNAcleavage in thymocytes without the involvement of endonucleases. Doesnot affect thiol proteases and metalloproteases Actinomycin D,Streptomyces Also acts as a competitive inhibitor of serine proteases;Classical sp. anti-neoplastic drug. Cytotoxic inducer of apoptosisagainst tumor cells. A DNA dependent inhibitor of RNA synthesis,actinomycin promotes induction of apoptosis by some specific stimuli,for example, TRAIL and Fas (CD95). Actinomycin D can also alleviate orblock the apoptotic process and decrease the cytotoxicity induced byseveral stimuli such as the dihydrofolate reductase inhibitoraminopterin and the prostaglandin derivative15-deoxy-D12,14-prostaglandin J2, thus it can have both pro andanti-apoptotic activities in some systems. According to one embodiment,said compound is not used as stabilizer according to the presentinvention. Aurintricarboxylic Acid Inhibitor of DNA topoisomerase IIBaicalein A cell-permeable flavone that inhibits the activity of 12-lipoxygenase (IC50 = 120 nM) and reverse transcriptase. Protectscortical neurons from β-amyloid induced toxicity. Reduces leukotrienebiosynthesis and inhibits the release of lysosomal enzymes. Alsoinhibits cellular Ca2+ uptake and mobilization, and adjuvant-inducedarthritis. Reported to inhibit microsomal lipid peroxidation by formingan iron-baicalein complex. Inhibits topoisomerase II and induces celldeath in hepatocellular carcinoma cell lines. Potentiates contractileresponses to nerve stimulation. Inhibits protein tyrosine kinase andPMA-stimulated protein kinase C Camptothecin, A cell-permeable DNAtopoisomerase I inhibitor. Exhibits anti- Camptotheca acuminata leukemicand antitumor properties. Induces apoptosis in HL-60 cells and mousethymocytes. Arrests cells at the G2/M phase Diisopropylfluorophosphateserine protease inhibitor Phenylmethylsulfonyl Fluoride Irreversibleinhibitor of serine proteases. Its mechanism of action is (PMSF)analogous to that of diisopropylfluorophosphate. PMSF causessulfonylation of the active-site serine residues. Also reported toinhibit internucleosomal DNA fragmentation in immature thymocytes. For arelated, more stable inhibitor, see AEBSF (−)-Huperzine A An inhibitorof AChE. Antagonist of NMDA receptors. Protects againstglutamate-mediated excitotoxicity. Razoxane Inhibits topoisomerase IIwithout inducing DNA strand breaks (topo II catalytic inhibitor).Suptopin-2 Suppressor of topoisomerase II inhibition. Reverses cellcycle arrest; bypass of checkpoint function. Has inherent fluorescenceand a distinct advantage in identification of molecule targets;effective concentraion in the μM range. 3. Enzymes 3.1. CaspasesApoptosis Inhibitor; 2-(p- Effects attributable to the inhibition ofcaspase-3 activation Methoxybenzyl)-3,4- pyrrolidinediol-3-acetatecIAP-1, Human, Recombinant, Recombinant, human cIAP-1 (amino acids1-618) fused to the E. coli peptide sequence MATVIDH10SSNG at theN-terminus and expressed in E. coli. cIAP is a member of the inhibitorof apoptosis family of proteins that inhibits proteolytic activity ofmature caspases by interaction of the BIR domain with the active caspaseCrmA, Recombinant CrmA (cowpox viral serpin cytokine response modifierA) is purified from E. coli transformed with a construct containing thefull-length coding region of the CrmA gene and 7 additional amino acidsthat do not affect the activity. CrmA is a natural inhibitor of humancaspase-1 and granzyme B, enzymes that are involved in apoptosis GroupIII Caspase Inhibitor I A potent, cell-permeable, and irreversibleinhibitor of Group III Peptide sequence: caspases (caspase-6, -8, -9,and -10), although more effective Ac-Ile-Glu-Pro-Asp-CHO, Ac- towardscaspases-6 and -8. Also inhibits caspase-1 and caspase- IEPD-CHO (SEQ IDNO: 3), 3. When using with purified native or recombinant enzyme,Caspase-8 inhibitor III pretreatment with an esterase is required.Kaempferol A cell-permeable phytoestrogen that inhibits topoisomerase I-catalyzed DNA religation in HL-60 cells. Offers protection againstAβ25-35-induced cell death in neonatal cortical neurons. Its protectiveeffects are comparable to that of estradiol. Blocks the Aβ-inducedactivation of caspase-2, -3, -8, and -9, and reduces NMDA-inducedneuronal apoptosis. Reported to be a potent inhibitor of monoamineoxidases. Acts as an inhibitor of COX-1 activity (IC50 = 180 μM), and oftranscriptional activation of COX-2 (IC50 < 15 μM Q-VD-OPH General,Pancaspase Boc-D(OMe)-FMK General, Pancaspase Z-D(OMe)E(OMe)VD(OMe)-Caspase 3, 7 FMK (SEQ ID NO: 4) Z-LE(OMe)TD(OMe)-FMK Caspase 8 (SEQ IDNO: 5) Z-YVAD(OMe)-FMK Caspase 1, 4 (SEQ ID NO: 6) Z-FA-FMK InhibitsCathepsin B Z-FF-FMK Cathepsin B, L Mu-PheHphe-FMK Cathepsin B, LZ-AE(OMe)VD(OMe)-FMK Caspase 10 (SEQ ID NO: 7) Z-ATAD(OMe)-FMK Caspase12 (SEQ ID NO: 8) Z-VK(Biotin)-D(OMe)-FMK General CaspaseZ-LE(OMe)VD(OMe)-FMK Caspase 4 (SEQ ID NO: 9) Z-VAM-FMK Antiviralpeptide inhibitor, Inhibits HRV2 and HRV14 4′-Azidocytidine HCVInhibitor Caspase-13 Inhibitor I A potent, reversible inhibitor ofcaspase-13 (ERICE). Peptide sequence: Ac-Leu-Glu-Glu-Asp-CHO (SEQ ID NO:10) Caspase-13 Inhibitor II A cell-permeable, irreversible inhibitor ofcaspase-13. When using Peptide sequence: with purified native orrecombinant enzyme, pretreatment with an Z-Leu-Glu(OMe)-Glu(OMe)-esterase is required. Asp(OMe)-FMK (SEQ ID NO: 11) Caspase-1 Inhibitor IA potent, specific, and reversible inhibitor of caspase-1 (Ki = 200Peptide sequence: pM for human recombinant caspase-1), caspase-4, andcaspase- Ac-Tyr-Val-Ala-Asp-CHO 5. Strongly inhibits anti-APO-1 inducedapoptosis in L929-APO-1 (SEQ ID NO: 12) cells. Caspase-1 Inhibitor I,Cell- A cell-permeable inhibitor of caspase-1 (ICE; Interleukin-1βPermeable Converting Enzyme), caspase-4, and caspase-5. The C-terminalPeptide sequence: YVAD-CHO sequence of this peptide is a highlyspecific, potent, Ac-Ala-Ala-Val-Ala-Leu-Leu- and reversible inhibitorof caspase-1 (Ki = 1 nM). The N-terminal Pro-Ala-Val-Leu-Leu-Ala-Leu-sequence (amino acid residues 1-16) corresponds to theLeu-Ala-Pro-Tyr-Val-Ala-Asp- hydrophobic region (h-region) of the signalpeptide of the Kaposi CHO fibroblast growth factor (K-FGF) and conferscell-permeability to (SEQ ID NO: 13) the peptide Caspase-1 Inhibitor IIA cell-permeable and irreversible inhibitor of caspase-1 (Ki = 760Peptide sequence: pM), caspase-4, and caspase-5. Inhibits Fas-mediatedapoptosis Ac-Tyr-Val-Ala-Asp-CMK and acidic sphingomyelinase activation(SEQ ID NO: 14) Caspase-1 Inhibitor IV A highly selective, competitive,cell-permeable, and irreversible Peptide sequence: inhibitor ofcaspase-1, caspase-4, and caspase-5. Inactivates theAc-Tyr-Val-Ala-Asp-AOM (AOM = enzyme with a rate limited by diffusionand is relatively inert toward 2,6-dimethylbenzoyloxymethyl otherbionucleophiles such as glutathione, making it an excellent ketone) (SEQID NO: 15) candidate for in vivo studies of enzyme inhibition Caspase-1Inhibitor V A potent inhibitor of caspase-1-like proteases. Blocksapoptotic cell Peptide sequence: death in human myeloid leukemia U937cells and blocks Z-Asp-CH2-DCB etoposide-induced DNA fragmentationCaspase-1 Inhibitor VI A potent, cell-permeable, and irreversibleinhibitor of caspase-1 Peptide sequence: (ICE), caspase-4, and caspase-5Z-Tyr-Val-Ala-Asp(OMe)—CH2F* (SEQ ID NO: 16) Caspase-2 Inhibitor I Acell-permeable and irreversible inhibitor of caspase-2 (ICH-1 Peptidesequence: Z-Val-Asp(OMe)-Val-Ala- Asp(OMe)—CH2F* (SEQ ID NO: 17)Caspase-2 Inhibitor II A reversible inhibitor of caspase-2 and caspase-3Peptide sequence: Ac-Leu-Asp-Glu-Ser-Asp-CHO (SEQ ID NO: 18) Caspase-3/7Inhibitor I A potent, cell-permeable, and specific, reversible inhibitorof Peptide sequence: caspase-3 (Ki = 60 nM) and caspase-7 (Ki = 170 nM).5-[(S)-(+)-2-(Methoxy- methyl)pyrrolidino]sulfonylisatin Caspase-3Inhibitor I A very potent, specific, and reversible inhibitor ofcaspase-3 (IC50 = Peptide sequence: 200 pM), caspase-6, caspase-7,caspase-8, and caspase-10. Ac-Asp-Glu-Val-Asp-CHO (SEQ ID NO: 19)Caspase-3 Inhibitor I, Cell- A cell-permeable inhibitor of caspase-3, aswell as caspase-6, Permeable caspase-7, caspase-8, and caspase-10. TheC-terminal DEVD- Peptide sequence: CHO sequence of this peptide is ahighly specific, potent, and Ac-Ala-Ala-Val-Ala-Leu-Leu- reversibleinhibitor of caspase-3 (Ki < 1 nM) that has also beenPro-Ala-Val-Leu-Leu-Ala-Leu- shown to strongly inhibit PARP cleavage incultured human Leu-Ala-Pro-Asp-Glu-Val-Asp- osteosarcoma cell extracts(IC50 = 200 pM). The N-terminal CHO (SEQ ID NO: 20) sequence (amino acidresidues 1-16) corresponds to the hydrophobic region (h-region) of thesignal peptide of Kaposi fibroblast growth factor (K-FGF) and conferscell-permeability to the peptide. A 5 mM (1 mg/100 μl) solution ofCaspase-3 Inhibitor I, Cell-permeable (Cat. No. 235427) in DMSO is alsoavailable. Caspase-3 Inhibitor II A potent, cell-permeable, andirreversible inhibitor of caspase-3 as Peptide sequence: well ascaspase-6, caspase-7, caspase-8, and caspase-10. WhenZ-Asp(OCH3)-Glu(OCH3)-Val- using with purified native or recombinantenzyme, pretreatment Asp(OCH3)-FMK with an esterase is required. A 5 mM(250 μg/75 μl) solution of Z- (SEQ ID NO: 21) DEVD-FMK (Cat. No. 264156)in DMSO is also available Caspase-3 Inhibitor III A potent,cell-permeable, and irreversible inhibitor of caspase-3 as Peptidesequence: well as caspase-6, caspase-7, caspase-8, and caspase-10Ac-Asp-Glu-Val-Asp-CMK (SEQ ID NO: 22) Caspase-3 Inhibitor IV A specificinhibitor of caspase-3. This tetrapeptide inhibitor has Peptidesequence. been used with the caspase-6 inhibitor Ac-VEID-CHO to dissectAc-Asp-Met-Gln-Asp-CHO the pathway of caspase activation inFas-stimulated Jurkat cells (SEQ ID NO: 23) Caspase-3 Inhibitor V Apotent, cell-permeable, and irreversible inhibitor of caspase-3, Peptidesequence: also recognizes caspase-1. When using with purified native orZ-Asp(OMe)-Gln-Met- recombinant enzyme, pre-treatment with an esteraseis required Asp(OMe)—CH2F* (SEQ ID NO: 24) Caspase-3 Inhibitor VII Acell-permeable, non-peptidyl pyrroloquinoline compound that Peptidesequence: acts as a potent, reversible, and non-competitive inhibitor of2-(4-Methyl-8-(morpholin-4- caspase-3 (IC50 = 23 nM) with 10-100-foldgreater selectivity. ylsulfonyl)-1,3-dioxo-1,3- Shown to display higheranti-apoptotic activity than Z-VAD-FMK dihydro-2H-pyrrolo[3,4- (Cat. No.627610) in a model of Staurosporine- (Cat. No. 569397)c]quinolin-2-yl)ethyl acetate induced apoptosis in human Jurkat T cells.Caspase-4 Inhibitor I A reversible caspase-4 inhibitor Peptide sequence:Ac-Leu-Glu-Val-Asp-CHO (SEQ ID NO: 25) Caspase-4 Inhibitor I, Cell- Apotent, cell-permeable, and reversible inhibitor of caspase-4. PermeableThe N-terminal sequence (amino acid residues 1-16) corresponds Peptidesequence: to the hydrophobic region of the signal peptide of Kaposifibroblast Ac-Ala-Ala-Val-Ala-Leu-Leu- growth factor and confers cellpermeability to the peptide. Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Leu-Glu-Val-Asp- CHO (SEQ ID NO: 26) Caspase-5 Inhibitor I Apotent, cell-permeable, and irreversible inhibitor of caspase-5. Peptidesequence: Strongly inhibits caspase-1. Also inhibits caspase-4 andcaspase-8 Z-Trp-Glu(OMe)-His- Asp(OMe)—CH2F* (SEQ ID NO: 27) Caspase-6Inhibitor I A cell-permeable, irreversible inhibitor of caspase-6. Whenusing Peptide sequence: with purified native or recombinant enzyme,pretreatment with an Z-Val-Glu(OMe)-Ile- esterase is requiredAsp(OMe)—CH2F* (SEQ ID NO: 28) Caspase-6 Inhibitor II, Cell- A potent,cell-permeable, and reversible inhibitor of caspase-6. The PermeableN-terminal sequence (amino acids 1-16) corresponds to the Peptidesequence: hydrophobic region of the signal peptide of Kaposi fibroblastAc-Ala-Ala-Val-Ala-Leu-Leu- growth factor and confers cell permeabilityto the peptide Pro-Ala-Val-Leu-Leu-Ala-Leu- Leu-Ala-Pro-Val-Glu-Ile-Asp-CHO (SEQ ID NO: 29) Caspase-8 Inhibitor I, Cell- A potent,cell-permeable, and reversible inhibitor of caspase-8 and PermeableGranzyme B. The N-terminal sequence (amino acids 1-16) Peptide sequence:corresponds to the hydrophobic region of the signal peptide ofAc-Ala-Ala-Val-Ala-Leu-Leu- Kaposi fibroblast growth factor and conferscell permeability to the Pro-Ala-Val-Leu-Leu-Ala-Leu- peptideLeu-Ala-Pro-Ile-Glu-Thr-Asp- CHO (SEQ ID NO: 30) Caspase-8 Inhibitor IIA potent, cell-permeable, and irreversible inhibitor of caspase-8Peptide sequence: and granzyme B. Effectively inhibits influenzavirus-induced Z-Ile-Glu(OMe)-Thr- apoptosis in HeLa cells. Also inhibitsgranzyme B. When using with Asp(OMe)—CH2F* purified native orrecombinant enzyme, pretreatment with an (SEQ ID NO: 31) esterase isrequired. A 5 mM (250 μg/76 μl) solution of Z-IETD- FMK (Cat. No.218840) in DMSO is also available. Caspase-9 Inhibitor I A potent,cell-permeable, and irreversible inhibitor of caspase-9. Peptidesequence: May also inhibit caspase-4 and caspase-5. When using withZ-Leu-Glu(OMe)-His- purified native or recombinant enzyme, pretreatmentwith an Asp(OMe)—CH2F* esterase is required. A 5 mM (250 μg/72 μl)solution of Z-LEHD- (SEQ ID NO: 32) FMK (Cat. No. 218841) in DMSO isalso available Caspase-9 Inhibitor II, Cell- A potent, cell-permeable,and reversible inhibitor of caspase-9. Permeable May also inhibitcaspase-4 and caspase-5. The N-terminal Peptide sequence: sequence(amino acids 1-16) corresponds to the hydrophobicAc-Ala-Ala-Val-Ala-Leu-Leu- region of the signal peptide of Kaposifibroblast growth factor and Pro-Ala-Val-Leu-Leu-Ala-Leu- confers cellpermeability to the peptide Leu-Ala-Pro-Leu-Glu-His-Asp- CHO (SEQ ID NO:33) Caspase-9 Inhibitor III A potent, irreversible inhibitor ofcaspase-9. Reported to reduce Peptide sequence: myocardial infarct sizeduring reperfusion (~70 nM). Ac-Leu-Glu-His-Asp-CMK (SEQ ID NO: 34)Caspase Inhibitor I A cell-permeable, irreversible, pan-caspaseinhibitor. Inhibits Fas- Peptide sequence: mediated apoptosis in Jurkatcells and staurosporine-induced cell Z-Val-Ala-Asp(OMe)—CH2F* death incorneal epithelial cells. When using with purified native or recombinantenzyme, pre-treatment with an esterase is required. Caspase Inhibitor IIA potent and reversible pan-caspase inhibitor. Peptide sequence:Ac-Val-Ala-Asp-CHO Caspase Inhibitor II, Cell- A cell-permeable,reversible pan-caspase inhibitor produced by Permeable attaching theN-terminal sequence (amino acids 1-16) of the Peptide sequence: Kaposifibroblast growth factor signaling peptide, which impartsAc-Ala-Ala-Val-Ala-Leu-Leu- cell-permeability to VAD peptide.Pro-Ala-Val-Leu-Leu-Ala-Leu- Leu-Ala-Pro-Val-Ala-Asp-CHO (SEQ ID NO: 35)Caspase Inhibitor III A cell-permeable, irreversible, broad-spectrumcaspase inhibitor. Peptide sequence: Boc-Asp(OMe)—CH2F* CaspaseInhibitor IV A general, irreversible caspase inhibitor. Peptidesequence: Boc-Asp(OBzl)-CMK Caspase Inhibitor VI An irreversible generalcaspase inhibitor. Useful for studies Peptide sequence: involvingrecombinant, isolated, and purified caspase enzymes. Z-Val-Ala-Asp-CH2F*Unlike Caspase Inhibitor I (Cat. No. 627610), this inhibitor does notrequire pretreatment with esterase for in vitro studies. A 10 mM (1mg/221 μl) solution of Caspase Inhibitor VI (Cat. No. 219011) in DMSO isalso available Caspase Inhibitor VIII A potent, reversible inhibitor ofcaspase-2 (Ki = 3.5 nM), caspase-3 Peptide sequence: (Ki = 1 nM) andcaspase-7 (Ki = 7.5 nM). Also serves as an Ac-Val-Asp-Val-Ala-Asp-CHOinhibitor of DRONC (Drosophila caspase), a glutamate/aspartate (SEQ IDNO: 36) protease. Caspase Inhibitor X A benzodioxane containing2,4-disubstituted thiazolo compound Peptide sequence: that acts as aselective, reversible and competitive inhibitor of BI-9B12 caspases (Ki= 4.3 μM, 6.2 μM and 2.7 μM for caspase-3, -7 and -8, respectively). Thebenzodioxane moiety is shown to fit in the ‘aspartate hole’ of thecaspases and possibly disrupt caspase-8 assisted cleavage of BID, aproapoptotic protein. Weakly affects the activity of anthrax lethalfactor, a metalloprotease, at ~20 μM Caspase-1 Inhibitors Including, butnot limited to Ac-N-Me-Tyr-Val-Ala-Asp-aldehyde (pseudo acid) (SEQ IDNO: 37) Ac-Trp-Glu-His-Asp-aldehyde (pseudo acid) (SEQ ID NO: 38)Ac-Tyr-Val-Ala-Asp-aldehyde (pseudo acid) (SEQ ID NO: 39)Ac-Tyr-Val-Ala-Asp-chloromethylketone (SEQ ID NO: 40)Ac-Tyr-Val-Ala-Asp-2,6-dimethylbenzoyloxymethylketone (SEQ ID NO: 41)Ac-Tyr-Val-Ala-Asp(OtBu)-aldehyde-dimethyl acetal (SEQ ID NO: 42)Ac-Tyr-Val-Lys-Asp-aldehyde (pseudo acid) (SEQ ID NO: 43)Ac-Tyr-Val-Lys(biotinyl)-Asp-2,6-dimethylbenzoyloxymethylketoneBiotinyl-Tyr-Val-Ala-Asp-chloromethylketoneBiotinyl-Val-Ala-DL-Asp-fluoromethylketoneFluorescein-6-carbonyl-Tyr-Val-Ala-DL-Asp(OMe)- fluoromethylketoneFluorescein-6-carbonyl-Val-Ala-DL-Asp(OMe)-fluoromethylketoneZ-Asp-2,6-dichlorobenzoyloxymethylketoneZ-Tyr-Val-Ala-Asp-chloromethylketone Z-Val-Ala-DL-Asp-fluoromethylketoneZ-Val-Ala-DL-Asp(OMe)-fluoromethylketone Caspase-2 Inhibitors Including,but not limited to Ac-Val-Asp-Val-Ala-Asp-aldehyde (pseudo acid) (SEQ IDNO: 44) Fluorescein-6-carbonyl-Val-Asp(OMe)-Val-Ala-DL-Asp(OMe)-fluoromethylketone (SEQ ID NO: 45)Z-Val-Asp(OMe)-Val-Ala-DL-Asp(OMe)-fluoromethylketone Caspase-3Precursor Protease Including, but not limited to InhibitorsAc-Glu-Ser-Met-Asp-aldehyde (pseudo acid) (SEQ ID NO: 46)Ac-Ile-Glu-Thr-Asp-aldehyde (pseudo acid) (SEQ ID NO: 47) Caspase-3Inhibitors Including, but not limited to Ac-Asp-Glu-Val-Asp-aldehyde(pseudo acid) (SEQ ID NO: 48) Ac-Asp-Met-Gln-Asp-aldehyde (pseudo acid)(SEQ ID NO: 49) Biotinyl-Asp-Glu-Val-Asp-aldehyde (pseudo acid)Caspase-3/7 Inhibitor IIFluorescein-6-carbonyl-Asp(OMe)-Glu(OMe)-Val-DL-Asp(OMe)-fluoromethylketone Z-Asp(OMe)-Gln-Met-DL-Asp(OMe)-fluoromethylketone(SEQ ID NO: 50) Z-Asp-Glu-Val-Asp-chloromethylketone (SEQ ID NO: 51)Z-Asp(OMe)-Glu(OMe)-Val-DL-Asp(OMe)-fluoromethylketone (SEQ ID NO: 52)Caspase-4 Inhibitors Including, but not limited toAc-Leu-Glu-Val-Asp-aldehyde (pseudo acid) (SEQ ID NO: 53)Z-Tyr-Val-Ala-DL-Asp-fluoromethylketone (SEQ ID NO: 54) Caspase-6Inhibitors Including, but not limited to Ac-Val-Glu-Ile-Asp-aldehyde(pseudo acid) (SEQ ID NO: 55)Fluorescein-6-carbonyl-Val-Glu(OMe)-Ile-DL-Asp(OMe)- fluoromethylketone(SEQ ID NO: 56) Z-Val-Glu(OMe)-Ile-DL-Asp(OMe)-fluoromethylketoneCaspase-8 Inhibitors Including, but not limited toAc-Ile-Glu-Pro-Asp-aldehyde (pseudo acid) (SEQ ID NO: 57)Boc-Ala-Glu-Val-Asp-aldehyde (pseudo acid) (SEQ ID NO: 58)Fluorescein-6-carbonyl-Ile-Glu(OMe)-Thr-DL-Asp(OMe)- fluoromethylketone(SEQ ID NO: 59) Fluorescein-6-carbonyl-Leu-Glu(OMe)-Thr-DL-Asp(OMe)-fluoromethylketone (SEQ ID NO: 60)Z-Ile-Glu(OMe)-Thr-DL-Asp(OMe)-fluoromethylketoneZ-Leu-Glu(OMe)-Thr-DL-Asp(OMe)-fluoromethylketone Z-LE(OMe)TD(OMe)-FMKCaspase-9 Inhibitors Including, but not limited toAc-Leu-Glu-His-Asp-aldehyde (pseudo acid) (SEQ ID NO: 61)Ac-Leu-Glu-His-Asp-chloromethylketone (SEQ ID NO: 62)Fluorescein-6-carbonyl-Leu-Glu(OMe)-His-DL-Asp(OMe)- fluoromethylketoneCaspase-10 Inhibitors Including, but not limited toFluorescein-6-carbonyl-Ala-Glu(OMe)-Val-DL-Asp(OMe)- fluoromethylketoneZ-Ala-Glu-Val-DL-Asp-fluoromethylketone (SEQ ID NO: 63) 3.2. CalpainCalpain Inhibitor III A potent, cell-permeable inhibitor of calpain Iand II (Ki = 8 nM). Peptide sequence: Reduces capsaicin-mediated celldeath in cultured dorsal root Z-Val-Phe—CHO ganglion. Reported to blockA23187-induced suppression of neurite outgrowth in isolated hippocampalpyramidal neurons. Exhibits neuroprotective effect in glutamate-inducedtoxicity. Calpain Inhibitor IV A potent, cell-permeable, andirreversible inhibitor of calpain II (k2 = Peptide sequence: 28,900M−1s−1). Also acts as an inhibitor of cathepsin L (k2 =Z-Leu-Leu-Tyr-CH2F 680,000 M−1s−1). Calpain Inhibitor V A potent,cell-permeable, and irreversible inhibitor of calpain Peptide sequence:Mu-Val-HPh—CH2F (Mu = morpholinoureidyl; HPh = homophenylalanyl)Ac-Leu-Leu-Nle-al Cell-permeable, peptide aldehyde inhibitor of calpainI (Ki = 190 nM), calpain II (Ki = 150 nM), cathepsin L (Ki = 0.5 nM) andother neutral cysteine proteases. Inhibits cell cycle progression atG1/S and metaphase/anaphase in CHO cells by inhibiting cyclin Bdegradation. Also stimulates HMG-CoA synthase transcription byinhibiting degradation of active SREBP-1 (sterol regulatoryelement-binding protein 1). Protects against neuronal damage caused byhypoxia and ischemia. Inhibits apoptosis in thymocytes andmetamyelocytes. Also prevents nitric oxide production by activatedmacrophages by interfering with the transcription of inducible nitricoxide synthase (iNOS; NOS II). Inhibits proteolytic degradation ofIkBalpha and IkBβ in RAW macrophages induced with LPS. It also prolongassociation of MHC class I molecules with the transporters associatedwith antigen processing Z-LLY-FMK Calpain N-Acetyl-Leu-Leu-Met Calpain IN-Acetyl-Leu-Leu-Nle-CHO Calpain I 3.3. others BAPTA/AMMembrane-permeable form of BAPTA. Can be loaded into a wide variety ofcells, where it is hydrolyzed by cytosolic esterases and is trappedintracellularly as the active chelator BAPTA. Prevents cocaine-inducedventricular fibrillations. Abolishes vitamin D3- induced increase inintracellular Ca2+. Induces inactivation of protein kinase C. Alsoinhibits thapsigargin-induced apoptosis in rat thymocytes. Granzyme BInhibitor I A weak inhibitor of the human and murine granzyme B. AlsoPeptide sequence: inhibits the apoptosis-related DNA fragmentation inlymphocytes Z-Ala-Ala-Asp-CH2Cl by fragmentin 2, a rat lymphocytegranule protease homologous to (SEQ ID NO: 64) granzyme B (ID50 = 300nM). Granzyme B Inhibitor II A potent, reversible inhibitor of granzymeB and caspase-8 (Ki = 1 Peptide sequence: nM). Also inhibits caspase-1(<6 nM), caspase-6 (5.6 nM), and Ac-Ile-Glu-Thr-Asp-CHO caspase-10 (27nM). (SEQ ID NO: 65) Granzyme B Inhibitor IV A reversible inhibitor ofgranzyme B and caspase-8 Peptide sequence: Ac-Ile-Glu-Pro-Asp-CHOLeupeptin, Hemisulfate, A reversible inhibitor of trypsin-like proteasesand cysteine Microbial proteases. Also known to inhibitactivation-induced programmed cell death and to restore defective immuneresponses of HIV+ donors N-Ethylmaleimide Sulfhydryl alkylating reagentthat inhibits H+-ATPase and suppresses the short circuit current (IC50 =22 μM) in pancreatic duct cells. Inactivates NADP-dependent isocitratedehydrogenase. Also a potent inhibitor of both Mg2+ andCa2+/Mg2+-stimulated DNA fragmentation in rat liver nuclei. Stimulatesarachidonic acid release through activation of PLA2 in endothelial cellsNα-Tosyl-Lys Chloromethyl Inhibits trypsin-like serine proteinases.Irreversibly inactivates Ketone, Hydrochloride (TLCK) trypsin withoutaffecting chymotrypsin. Prevents nitric oxide production by activatedmacrophages by interfering with transcription of the iNOS gene. Blockscell-cell adhesion and binding of HIV-1 virus to the target cells. Inmacrophages, blocks nitric oxide synthase induced by interferon-γ andlipopolysaccharides (EC50 = 80 μM). Prevents endonucleolysisaccompanying apoptotic death of HL-60 leukemia cells and normalthymocytes Omi/HtrA2 Protease Inhibitor, A cell-permeablefurfurylidine-thiobarbituric acid compound that Ucf-101 acts as apotent, specific, competitive, and reversible inhibitor of thepro-apoptotic, heat-inducible, mitochondrial serine protease Omi/HtrA2(IC50 = 9.5 μM for His-Omi134-458). Shows very little activity againstvarious other serine proteases tested (IC50 ≥ 200 μM). Reported to blockOmi/HtrA2 induced cell death in caspase-9 (−/−) null fibroblasts.Phenylarsine Oxide A membrane-permeable protein tyrosine phosphataseinhibitor (IC50 = 18 μM). Stimulates 2-deoxyglucose transport ininsulin- resistant human skeletal muscle and activates p56lck proteintyrosine kinase. Blocks TNF-α-dependent activation of NF-κB in humanmyeloid ML-1a cells. PAO inhibits the protease activities of recombinanthuman caspases as well as endogenous caspases that are active inextracts of pre-apoptotic chicken DU249 cells (S/M extracts).Phorbol-12,13-dibutyrate Strong irritant for mouse skin, but onlymoderately active as a tumor promoter. Activates protein kinase C.Stimulates the phosphorylation of Na+, K+-ATPase, thereby inhibiting itsactivity. Promotes the expression of inducible NOS in culturedhepatocytes. Commonly used in binding studies or in applicationsrequiring high concentrations of phorbol compounds. Hypericin InhibitsPKC, CKII, MAP Kinase, Insulin R, EGFR, PI-3 Kinase and also noted topossess antiviral activity. Butyrolactone I A cell-permeable and highlyselective inhibitor of cyclin-dependent protein kinases (Cdks) thatinhibits cell cycle progression at the G1/S and G2/M transitions.Inhibits p34cdk1/cyclinB (Cdk1; IC50 = 680 nM). Also selectivelyinhibits Cdk2 and Cdk5 kinases. Has little effect on casein kinase I,casein kinase II, EGF receptor kinase, MAP kinase, PKA, and PKC. Shownto prevent the phosphorylation of retinoblastoma protein and H1 histone.Also blocks Fas-induced apoptosis in HL-60 cells and shows antitumoreffects on human lung cancer cell lines Nilotinib SpezifischerBCR-ABL-Tyrosinkinase-Inhibitor Quercetin(Sophoretin) Quercetin is aPI3K and PKC inhibitor with IC50 of 3.8 μM and 15 μg/ml. It stronglyabrogated PI3K and Src kinases, mildly inhibited Akt1/2, and slightlyaffected PKC, p38 and ERK1/2. [1][2] Quercetin is a naturally-occurringpolar auxin transport inhibitor with IC50 of 0.8, 16.7, 6.1, 11.36 μMfor the inhibition of LDH % release, the inhibition of TNF-inducedPMN-EC adhesion, TNF- induced inhibition of DNA synthesis andproliferation. It is a type of plant-based chemical, or phytochemical,known as a flavonol and a plant-derived flavonoid found in fruits,vegetables, leaves and grains. It also may be used as an ingredient insupplements, beverages or foods. In several studies, it may have anti-inflammatory and antioxidant properties, and it is being investigatedfor a wide range of potential health benefits

EXAMPLES

In the following examples, materials and methods of the presentinvention are provided. It should be understood that these examples arefor illustrative purpose only and are not to be construed as limitingthis invention in any manner.

I. Materials and Methods

A test system was designed, wherein cell-containing biological samples,here whole blood samples, were incubated at room temperature (RT) for upto 6 or 7 days. Therein, the sample stabilizing properties of theadditives of the present invention were tested on day 0, day 3 and day6/7 the samples. The samples were processed according to the followingprotocols, where applicable (for details, see also the specific examplesin the results section):

1. Measurement of Blood Cell Integrity by Fluorescence Activated CellSorting (FACS)

1.1. Lysis of Red Blood Cells

-   -   Transfer of 2 ml blood sample into a fresh 15 ml Falcon tube    -   Addition of 5-fold Buffer EL (QIAGEN)    -   Inverting of the sample (10×)    -   Incubation on ice (10 min.)    -   Centrifugation for 10 min. @400×g and 4° C.    -   Discard of the supernatant    -   Addition of 2-fold Buffer EL (QIAGEN) to the white blood cell        pellet    -   Resolution of the pellet in Buffer EL (QIAGEN) by slight        vortexing    -   Centrifugation for 10 min @ 400×g    -   Discard of the supernatant    -   Addition of 500 μl FACS Flow (Becton, Dickinson Plymouth, UK) to        the white blood cell pellet    -   Resolution of the pellet in FACS Flow by slight vortexing    -   Transfer of 1 ml FACS Flow into a fresh FACS tube    -   Transfer of 100 μl of the resolved pellet into a FACS tube

Red blood cells are lysed because otherwise, the decisive cellpopulations (which can release e.g. genomic DNA) are not distinguishablein the FACS analysis due to the high amount of red blood cells.

1.2. Measurement of Cell Integrity by Flow Cytometry

The measurement was performed according to manufacturer's instruction(FACSCalibur; Becton, Dickinson Plymouth, UK).

2. Separation of Blood Plasma

To separate the blood plasma from the whole blood, the blood sampleswere centrifuged for 15 min at 5000 rpm, and the obtained plasma sampleswere again centrifuged for 10 min at 16.000×g at 4° C.

The resulting blood plasma was used for isolating the nucleic acidscontained therein.

3. Nucleic Acid Purification

The circulating, extracellular nucleic acids were purified from theobtained plasma samples using the QIAamp® Circulating NA Kit (accordingto the handbook). In brief:

-   -   10 ml sample input;    -   lysis: 1 ml Proteinase K and 8 ml Buffer ACL (QIAGEN)    -   binding: 18 ml Buffer ACB (QIAGEN)    -   wash-steps: unchanged and according to handbook    -   elution in 60 μl Buffer AVE (QIAGEN)        4. Analysis of the Eluates

The eluates obtained according to 3. were stored at −20° C. till allsamples (including day 6/7 samples) were purified. Afterwards, eluatesof the same condition were pooled and treated as follows:

4.1. Measurement of the blood cell stability/DNA release by thedetermination of DNA size distribution using a chip gel electrophoresis(2100 Bioanalyzer; Agilent Technologies; Inc., USA) according tomanufacturer's instruction (see handbook Agilent DNA 7500 and DNA 12000Kit Guide), but 1.5 μl instead of 1 μl sample were transferred to thewells.

4.2. DNA quantification with a real time PCR assay, sensitive for DNAdegradation (target: 500 and 66 bp long ribosomal 18S DNA codingsequences).

The DNA duplex assay was carried out according to the QuantiTect®Multiplex PCR handbook (Qiagen) with the following adaptions:

-   -   Primer concentration was up scaled from 8 μM to 16 μM.    -   Annealing/extension step was extended from 1 to 2 min. (samples        were diluted 1:10 before amplification)

4.3. RNA detection using real time PCR assays, sensitive for variationsin circulating cell-free RNA levels (target: 18S rRNA, IL8, c-fos andp53). The RNA assays were carried out according to the conditionsdescribed in Tables 2 to 4.

TABLE 2 shows compositions of PCR reagents and cycling conditions of thep53 mRNA one step real time PCR. TaqMan MasterMix (MM) single master-mixcomponent reaction (x-fold) c p53 FAM-BHQ + HEX-BHQ x-fach 1 x 182 1 x20.00 mastermix/reaction RNA 5.000 / var. 5 μl RNA A. dest (PCR grade)3.813 693.9 / 25.00 μl reaction volume 2x QuantiTect Probe RT-PCRMasterMix (Puffer) 12.500 2275.0 1 x forw. primer (20 μM) 0.500 91.0 400nM rev. primer (20 μM) 0.500 91.0 400 nM probe (20 μM) 0.313 56.9 250 nMRNAsin (40 U/μl; Promega) 0.125 22.8 0.2 U/μl MgCl2 (25 mM) 2.000 364.06 mM QuantiTect RT-PCR Mix (Enzym Mix) 0.250 45.5 U/μl Reaction volume[μl] 25.000 3640.0 Cycling: 30 min 50° C. 15 min 95° C. 40 cycles 15seq. 95° C.

TABLE 3 shows compositions of PCR reagents and cycling conditions of theIL8 mRNA one step real time PCR. single master-mix final componentreaction (x-fold) konc. IL8 FAM-BHQ x-fold 1 x 106 1 x 20.00 μlmastermix/reaction RNA 5.000 / var. 5 μl RNA A. dest (PCR grade) 3.751397.6 / 25.00 μl reaction volume 2x QuantiTect Probe RT-PCR MasterMix(Puffer) 12.500 1325.0 1 x forw. primer (40 μM) 0.562 59.6 900 nM rev.primer (40 μM) 0.562 59.6 900 nM probe (20 μM) 0.250 26.5 200 nM RNAsin(40 U/μl; Promega) 0.125 13.3 0.2 U/μl MgCl2 (25 mM) 2.000 212.0 6 mMQuantiTect RT-PCR Mix (Enzym Mix) 0.250 26.5 U/μl Reaction volume [μl]25.000 2120.0 Cycling: 30 min 50° C. 15 min 95° C. 40 cycles 15 seq. 95°C. 1 min. 60° C.

TABLE 4 shows compositions of PCR reagents and cycling conditions of thec-fos mRNA/18S rRNA duplex real time PCR. for single MM Chemicals CFOSreaction x-fold FAM-JOE x-fold 1x 220 17.6 μl mastermix/ reactionTemplate 2.4 / 2.4 μl RNA A. dest 1.00 220 20 μl reaction 2x QuantiTecMastermix 10 2200 volume forw. primer (20 μM); c-fos 0.900 198 rev.primer (20 μM); c-fos 0.900 198 probe (10 μM); c-fos 0.500 110 forw.primer (10 μM); 18 S 0.800 176 rev. primer (10 μM); 18 S 0.800 176 probe(10 μM); 18 S 0.800 176 RNasin (40 U/μl; Promega) 0.100 22 MgCl2 (25 mM)1.6 352 QuantiTect RT-PCR Mix 0.2 44 (Enzym Mix) reaction volume [μl]20.0 3872 Cycling: 30 min 50° C. 15 min 95° C. 40 cycles 15 sec. 95° C.1.30 min. 60° C.

TABLE 5summarizes the information of the used DNA target sequences detected in 4.2 and 4.3 amplicon sequence length target description positionsize [bp] position 5′-3′ [nt] dye 18 S human p12- 66 forwardGCCGCTAGAGGT 22 5′ Cy5- ribosomal region GAAATTCTTG BHQ 3′ DNA of(SEQ ID NO: 66) chromo- reverse CATTCTTGGCAA 21 some ATGCTTTCG 13, 14,(SEQ ID NO: 67) 15, 21, probe ACCGGCGCAAGA 21 22 CGGACCAGA(SEQ ID NO: 68) 18 S human p12- 500 forward GTCGCTCGCTCC 22 5′ FAM-ribosomal region TCTCCTACTT BHQ 3′ DNA of (SEQ ID NO: 69) chromo-reverse GGCTGCTGGCAC 19 some CAGACTT 13, 14, (SEQ ID NO: 72) 15, 21,probe CTAATACATGCC 25 22 GACGGGCGCTGA C (SEQ ID NO: 71)II. Performed Experiments and Results

Subsequently, the details on the performed experiments are explained.Details to the methods used in the examples were described above underI.

Example 1 Stabilization by the Addition of a Caspase-Inhibitor

Two different oligopeptides, Q-VD-OPh and Z-Val-Ala-Asp(OMe)-FMK actingas broad spectrum caspase-inhibitors, were tested:

TABLE 6 Tested caspase inhibitors inhibitor moleculare name weightsolubility structure Q-VD-OPH 513.49 20 mM, add 97 μl DMSO 10 mM, add194 μl DMSO  5 mM, add 388 μl DMSO

Z-Val-Ala- Asp(Ome)- FMK 467.49 20 mM, add 107 μl DMSO 10 mM, add 214 μlDMSO  5 mM, add 428 μl DMSO

Each tested caspase inhibitor was added to whole blood samples (20 μMend concentration in 10 ml blood; blood was collected into VacutainerK2E Tubes; BD). The whole blood sample was processed as described insection I, see 2. (plasma preparation) and 3. (nucleic acid isolation).

Results of the Chip Gel Electrophoresis

The eluted circulating cell-free DNA was separated by size using chipgel electrophoresis (for details on the method see above, I, 4.1). FIG.1a shows the obtained results. The DMSO control and the K2E blood (nottreated according to the teachings of the present invention) show thesame ladder-like pattern of bands. This pattern occurs in samples whereapoptosis takes place. During apoptosis, endonucleases degrade genomicDNA at inter-nucleosomal linker regions and produce DNA fragments ofcirca 180 bp or multiples of 180 bp. Thus, apoptosis occurs in sampleswhich show a clear ladder-like pattern. Furthermore, the strength(darkness) of the pattern is decisive. The darker the bands, the moregenomic DNA was released from the cells and thus contaminates theextracellular nucleic acid population.

FIG. 1 a) shows that the DMSO control and the K2E blood samples show astrong ladder-like pattern already on day 3, which becomes even strongeron day 7. Thus, genomic DNA was released from the cells contained in thesample and was also degraded. This released and degraded DNAcontaminates the cell-free nucleic acids contained in the sample. Hence,no acceptable stabilisation is achieved with these samples.

In contrast, whole blood samples treated with Z-Val-Ala-Asp(OMe)-FMKshow a reduced ladder-like pattern in particular on day 7 compared tothe controls, indicating an inhibition of the release of genomic DNA,respectively genomic DNA fragmentation caused by apoptosis. This effectis confirmed by the results shown in FIG. 1 b) (see below). The effectis even more prominent in the blood samples treated with Q-VD-OPh, whichshow significantly reduced ladder-like patterns already on day 3 and day7. Thus, the release and degradation of genomic DNA is effectivelyprevented, respectively reduced by the addition of the caspase inhibitorQ-VD-OPh.

Results of the DNA Quantification

The eluted circulating cell-free DNA was also quantified with the realtime PCR assay that is sensitive for DNA degradation (for details on themethod see above, I, 4.2). FIG. 1 b) shows the effect of the testedcaspase-inhibitors on the stabilisation of the extracellular nucleicacid population (18S DNA duplex assay) within 7 days of storage at RT,here the increase in DNA.

Detection of ribosomal 18S DNA by quantitative real-time PCR, makes itpossible to calculate the x-fold increase of DNA from day 0 to day 3 or7 (calculation: division of day 3 (or 7) copies by day 0 copies).Surprisingly, the results shown in FIG. 1 b) demonstrate a reducedincrease of DNA when a caspase-inhibitor, especially Q-VD-OPh, was addedto whole blood samples. The stabilising effect of Z-Val-Ala-Asp(OMe)-FMKcompared to the standard samples was more prominent on day 7, therebyconfirming the results shown in FIG. 1 a).

SUMMARY

Summarizing the results of the real time PCR and the gelelectrophoresis, it was demonstrated that the addition of Q-VD-OPh orZ-Val-Ala-Asp(OMe)-FMK inhibits DNA fragmentation and furthermore,reduces the release of genomic DNA into blood plasma. Thus, adding acaspase inhibitor to whole blood is effective in stabilising the sampleand in particular the extracellular nucleic acid population even at roomtemperature. Thus, using the stabilisation method according to thepresent invention, allows to ship whole blood samples even at roomtemperature without jeopardizing the quality of the sample. Tocompletely prevent release of genomic DNA also during longer storageperiods, the concentration of Q-VD-OPh may also be increased.

Example 2 Influence of Lower Concentrations of Caspase-InhibitorQ-VD-OPh on Blood Stability

In this example, lower concentrations of the caspase inhibitor Q-VD-OPhwas tested in combination with glucose, wherein the glucose was added ascombination partner to support that the blood cells stay alive (bypreventing cell damage). 21.4 mM glucose and 4 μM, 1 μM or no Q-VD-OPhwere added to 10 ml blood drawn into BD Vacutainer tubes and stored forup to 7 days at room temperature. The whole blood sample was processedas described in section I, see 2. (plasma preparation) and 3. (nucleicacid isolation).

Results of the Chip Gel Electrophoresis

The eluted DNA was separated by size using chip gel electrophoresis (fordetails on the method see above, I, 4.1). FIG. 2a shows that compared tothe control samples, wherein no caspase inhibitor was added, already 1μM caspase inhibitor significantly reduced the genomic DNArelease/fragmentation on day 7. The effect is improved if 4 μM caspaseinhibitor is used. Thus, already very low concentrations of the caspaseinhibitor are effective in stabilising the blood sample, in particularwhen combined with a carbohydrate.

Results of the DNA Quantification

FIG. 2b shows the effects of the tested concentrations of thecaspase-inhibitor Q-VD-OPh in combination with 21 mM glucose on theincrease of genomic DNA in the plasma (18S DNA duplex assay) within 7days of storage at RT. The addition of Q-VD-OPh in combination withglucose significantly reduces the release of genomic DNA into plasma.FIG. 2b shows only a minor increase of genomic DNA within 7 days ofstorage even if only 1 μM Q-VD-OPH was added to the whole blood samplefor stabilisation. The addition of 4 μM Q-VD-OPh inhibits the release ofgenomic DNA to plasma to a maximum of a 4-fold increase. In contrast,drawing whole blood in K2E Tubes without stabilisation according to thepresent invention leads to approximately 40-fold increase of DNA inplasma.

Thus, also FIG. 2 b) confirms that the caspase inhibitor has astabilisation effect on whole blood even at low concentrations.

Example 3 Stabilizing Blood Cells by Osmotic Effects

Surprisingly it was also found by the inventors that blood cells can bestabilized by adding a reagent that acts as a hypertonic medium in wholeblood. Generating a hypertonic medium by the addition of, for example,hydroxylated organic compound(s) to whole blood results in a slightrelease of water from the contained blood cells and results in increasedstability by cell shrinking. It is assumed that said cell shrinkingstabilises the cells against mechanical forces.

Dihydroxyacetone (DHA) is an intermediate product of the fructosemetabolism and its phosphate form dihydroxyacetone phosphate (DHAP) ispart of the glycolysis. DHA was tested as hypertonic agent. Addition ofthis reagent sensitively forces blood cells to shrink without damagingthem. DHA was first dissolved in PBS (purchased from SIGMA-Aldrich Kat.No: D8537) or 3×MOPS (diluted from 1 litre of 10×MOPS: 200 mM MOPS; 50mM NaAc, 10 mM EDTA; pH 5; assuming that an acid medium also stabilizesccf RNA) obtaining 4.2M solved DHA. Then 2 ml of 4.2M DHA dissolved inBuffer PBS or buffer 3×MOPS were added to 10 ml of blood to obtain afinal concentration of 0.7M DHA in whole blood. The two differentsolvents of DHA were compared to PAXgene® Blood DNA tubes (QIAGEN), astate-of-the-art blood collection tube for DNA stabilization.

Results of the FACS Analysis

The blood cell integrity was analysed using FACS (for details on themethod see above, I, 1). FIG. 3 shows the blood cell integrity measuredby flow cytometry. The Dot-Plots visualize three different cellpopulations: granulocytes (1), monocytes (2) and lymphocytes (3). Thecloud (4) in the lower left field of the plot represents the debris,mainly generated by the lysis of erythrocytes.

The results in FIG. 3 show that blood cells collected and stored inPAXgene® Blood DNA tubes are not distinguishable from each other and thedebris on day 6 of storage. The addition of DHA enables adifferentiation of the subpopulations of blood cells on day 6 of storageeven though these cells become smaller as a result of the cellshrinking. This indicates that the cells contained in the sample werestabilised by the addition of DHA.

Results of the Chip Gel Electrophoresis

The results presented in FIG. 4a also shows a stabilisation of the bloodsamples by the addition of DHA, because the release of genomic DNA issignificantly lower with the DHA treated samples than in samples storedin PAXgene® Blood DNA tubes. Furthermore, as is evident from FIG. 4a ,DHA-stabilized samples do not show ladder-like degradation patternsuggesting that apoptosis, respectively a degradation of DNA isefficiently prevented.

Results of the DNA Quantification

FIG. 4b shows the effect of DHA on the increase of DNA (18S DNA duplexassay) within 6 days of storage at RT. DHA dissolved in 3×MOPS providedthe best results, because the level of ribosomal 18S DNA seems to remainconstant till day 3 of storage.

The division of short amplicon copy number by long amplicon copy number(66 by/500 bp) indicates whether the amount of detected short or longamplicons changes over time in a similar way. A decrease of this ratioimplies a stronger release of longer rather than of shorter DNAmolecules and can be interpreted as release of high molecular weightgenomic DNA from blood cells. The diagram shown in FIG. 4b indicates therelease of genomic DNA for all three conditions. The results show thatthe presence of DHA slows this process down. Thus, also this experimentshows that the addition of DHA to whole EDTA blood stabilizes bloodcells and hence preserves the ccfDNA population in the cell-free plasmafraction and avoids contaminations with DNA released from the cellscontained in the sample e.g. due to mechanical breakup.

Example 4 Testing Different Concentrations of Dihydroxyacetone

In this example, the stabilising effect of different concentrations ofDHA (0.7M, 0.5M and 0.2M) was tested.

Results of the FACS Analysis

FIG. 5 shows the blood cell integrity measured by flow cytometry. TheDot-Plots visualize three different cell populations: granulocytes (1),monocytes (2) and lymphocytes (3). The cloud in the lower left field ofthe plot represents the debris, mainly caused by the lysis oferythrocytes.

Due to the addition of DHA to whole blood the different cell populationscan be distinguished even on day 6 of storage regardless of the DHAconcentration. Although the results of the flow cytometry analysis (FIG.5) do not show differences in cell integrity between the differentconcentrations of DHA

Results of the Chip Gel Electrophoresis

The results presented in FIG. 6a also show a stabilisation of the bloodsamples by the addition of the different concentrations of DHA, becausethe release of genomic DNA and the degradation of the DNA is efficientlyprevented.

Results of the DNA Quantification

FIG. 6b shows the effect of different DHA concentrations on the increaseof DNA (18S DNA duplex assay) within 6 days of storage at RT. As shownin FIG. 6 b, 0.5M DHA in whole blood prevents most efficiently therelease of genomic DNA. Furthermore, the ratio of short to long ampliconcopy numbers stays constant for up to 3 days and only decreases slightlytill day 6. These results demonstrate the remarkable effect of thehypertonic agent DHA on the stabilisation of whole blood.

Example 5 Combination of an Apoptosis Inhibitor, an Osmotically ActiveCompound and an Anticoagulant

An increase of EDTA in blood collection tubes inhibits micro- andmacroclotting as it is known for PAXgene® Blood DNA tubes. Hence, higherconcentrations of EDTA may support stabilization of blood cells andextracellular nucleic acids in plasma. Furthermore, the experimentspresented above show an inhibitory effect of the caspase inhibitor, inparticular Q-VD-OPh, and the osmotically active compound DHA on bloodcell damage and in particular show that an increase of genomic DNA, inparticular fragmented genomic DNA, in the extracellular nucleic acidpopulation is efficiently reduced. Surprisingly, the caspase inhibitorstested also prevented/inhibited the leakage of genomic DNA into thecell-free (plasma) fraction. Hence, the combination of these reagentsresults in an improved stabilization of extracellular nucleic acids, inparticular extracellular DNA, in whole blood that lasts at least for 6days, and furthermore, results in an efficient stabilization of bloodcells, thereby preventing the release of genomic DNA, what otherwisewould result in a dilution of the natural extracellular nucleic acidlevel in plasma.

In this example, DHA was dissolved in 2 ml 3×MOPS (3M DHA in 2 ml3×MOPS), 50 mg K₂EDTA and 2.4 μl of 5 nM Q-VD-OPh were added and thentransferred into 10 ml whole blood, that was collected in K2E Tubes.Plasma samples were centrifuged for 10 min at 16.000×g, 4° C. and thenpurified using the QIAamp® Circulating NA Kit (Qiagen) (details aredescribed above in section I).

Results of the FACS Analysis

FIG. 7a shows the blood cell integrity measured by flow cytometry. TheDot-Plots visualize three different cell populations: granulocytes (1),monocytes (2) and lymphocytes (3). The cloud in the lower left field ofthe plot represents the debris, mainly caused by the lysis of remainingerythrocytes.

The addition of the caspase inhibitor, the hypertonic agent and thecomplexing agent to whole blood resulted in a distinguishable pattern ofblood cell populations after 6 days of storage. Thus, the cellscontained in the blood sample were efficiently stabilised.

Results of the DNA Quantification

FIG. 7b shows the effect of the combination of EDTA, DHA and thecaspase-inhibitor Q-VD-OPH on the increase of DNA (18S DNA duplex assay)within 6 days of storage at RT. The results indicate that thecombination of EDTA, DHA and Q-VD-OPH leads to a remarkably strongstabilization of extracellular DNA in plasma (level of measured 18S rDNAremains constant till day 6) and to a strong prevention of the releaseof genomic DNA from blood cells (ratio of short to long amplicon copynumbers remains constant) till day 3 of storage. Only a slight increaseof genomic DNA into plasma becomes visible between day 3 and day 6 ofstorage.

Thus, the tested combination of stabilising agents is particularlyefficient in stabilising whole blood samples.

Example 6 Effect of an Apoptosis Inhibitor, an Osmotically ActiveCompound and a Preventing Agent on Free Circulating RNA in Whole Blood

As a combination of K₂EDTA, Q-VD-OPh and DHA showed remarkablestabilizing effects on free circulating DNA and the integrity of bloodcells in whole blood, the stabilising capacities of these agents on freecirculating RNA was also analysed. To preserve a constant level of freecirculating RNA in plasma (as present when collecting the blood), thestabilizing reagent(s) should not only protect RNAs from degradation andprevent the release of RNAs from decaying blood cells, but should alsoinhibit the metabolic pathways, respectively have the effect thatchanges in the metabolic pathway do not affect the extracellular RNAplasma level, respectively should reduce respective effects. Henceexperiment 5 was repeated and the level of mRNA was measured by realtime RT-PCR.

FIG. 8 shows the effect of the combination of EDTA, DHA and the testedcaspase-inhibitor on the transcript level in plasma within 6 days ofstorage. In order to measure variations in RNA levels, target mRNAs werereferred to as reference target (18S rRNA) by calculating a ΔCt betweenp53, IL8 or c-fos and the internal standard (18S rRNA). Subtracting theΔCt of day 3 or 6 samples with the ΔCt of day 0 samples defines the 4ΔCt visualizing a relative decrease (− values) or increase (+ values) ofmRNA transcript levels. IL8 and c-fos are genes whose transcription isinduced after blood draw. Therefore, transcript levels of these targetswould rise dramatically when cells release their contents; the additionof the stabilizing solution according to the preferred embodiment ofpresent invention (combination of elevated EDTA, dihydroxyacetone,caspase inhibitor Q-VD-OPh) strongly prevents nucleic acid release fromblood cells till day 3 of storage. But the data in the diagram aboveshow—surprisingly—no significant increase of c-fos and IL8 mRNA till day6 of storage. Thus, apparently the stabilization prevents thedegradation of RNA (p53) and the release of mRNA (IL8/c-fos)

The transcription of p53 is repressed during continued metabolism afterblood draw and, hence, a degradation or down-regulation of p53 mRNAwould result in a decrease of (−)ΔΔcts. However, the results show thatthe tested QGN stabilisation solution prevents the p53 mRNA-level frombeing degraded during whole blood storage for up to 6 days.

This experiment demonstrated that the addition of a combination ofelevated EDTA, dihydroxyacetone, caspase inhibitor Q-VD-OPh to freshlydrawn whole blood acts to preserve the circulating plasma mRNApopulation which was present at the time of blood draw, reducingmRNA-specific changes in mRNA concentration. This is of particularimportance for the analysis of circulating mRNA in plasma, e.g., foridentification and characterization of potential tumor-specific mRNAspecies. Such studies require that the mRNA population in plasma remainssubstantially unchanged between blood draw and nucleic acid extractionand analysis.

Example 7 Stabilisation by the Addition of Dimethylacetamide (DMAA)

Two different concentrations of DMAA along with K₂EDTA were tested andcompared to EDTA alone (K2E BD; 18 mg K₂EDTA).

DMAA was added to replicates of whole blood samples (0.75% and 1.5% endconcentration in 10 ml blood; blood was collected into Vacutainer K2ETubes; BD).

Blood samples were incubated for up to 6 days at room temperature. Onday 0, 3 and 6, whole blood samples were centrifuged at 1912×g for 15min at room temperature, followed by a centrifugation of the plasmasamples at 16.000×g for 10 min at 4° C. 1 ml of the sample input wasused for DNA isolation following the protocol described in the materials& methods section. DNA was eluted in 80 μl EB buffer and quantified withthe RT PCR assay described in I, 4.2.

Results of the DNA Quantification

FIG. 11 shows the effects of the tested concentrations of DMAA on theincrease of genomic DNA in the plasma. Addition of DMAA significantlyreduces the release of genomic DNA into plasma. The more DMAA is addedto whole blood, the less DNA is released. Only a minor increase ofcell-free DNA within 6 days of storage was observed if 1.5% DMAA wasadded to the whole blood sample. Furthermore, as the addition of 1.5%DMAA stabilizes cell-free DNA levels in whole blood samples moreefficiently than 0.75% and the ratio of short to long measured 18S DNAcopies decreases from day 0 to day 6, higher DMAA concentrations of than1.5% can result in more efficient stabilization effects.

In summary, the addition of DMAA reduces the release of genomic DNA intoblood plasma. Thus, adding DMAA to a blood sample is effective instabilising the sample even at room temperature.

Example 8 Influence of Sugar Alcohols on Preserving the ccfDNA Status inWhole Blood

10 ml whole blood samples of two donors were first collected in BDVacutainer K2E-EDTA (4.45 mM EDTA=Reference). Afterwards, 2 ml of thefollowing stabilization solutions were added (given concentrationsrepresent final concentration in stabilized blood solution):

Stabilization Inositol Maltitol Mannitol Sorbitol DHA None Solution (M)(M) (M) (M) (M) (K2E) 1 0.1 2 0.05 3 0.01 4 0.1 5 0.05 6 0.01 7 0.1 80.05 9 0.01 10 0.1 11 0.05 12 0.01 13 0.5 14 X

The respectively stabilized samples were incubated at room temperaturefor up to six days. On day 0, day 3 and day 6, replicates were processedas follows. The samples were centrifuged at 3.000 rpm for 10 minutes atroom temperature in order to collect plasma. The collected plasma wascentrifuged at 16,000×g for 10 minutes at 4° C. The cleared plasmafraction was collected and the extracellular nucleic acids were isolatedusing the QIAamp Circulating nucleic acid kit (1 ml input material, 60μl elution volume). The results are shown in relative change compared tothe test time point 0 days (day X copies/day 0 copies) in FIG. 10.Values that are close to 1 imply preserved levels of ccfDNA. The higherthe value, the less stabilization is achieved. From the sugar alcoholstested, very good results were achieved with DHA (0.5 M). Here, thelowest increase of ccfDNA levels in the plasma fraction was observed.Other suitable alternatives are inositol in concentrations of forexample 0.05 M and maltitol in concentrations ≤0.01M. Furthermore,stabilization effects over 3 days were also seen with mannitol.

Example 9 Influence of DMAA, DHA and Glycine on ccfDNA Level

10 ml whole blood samples of three donors were first collected in BDVacutainer K2E-EDTA (4.45 mM EDTA=reference). Afterwards, 2 ml of thefollowing solutions were added (given concentrations represent finalconcentration in stabilized blood solution):

OPH Stabilization DHA DMAA (caspase None Solution (M) (%) EDTAinhibitor) (K2E) 1 0.5 2 0.1 3 0.05 4 (QGN mixture) 0.5 14 mM 1 μM 5 1%14 mM 1 μM 6 3% 14 mM 1 μM 7 5% 14 mM 1 μM 8 X

The samples were processed as described in example 8. The results aswell as the test conditions are shown in FIG. 11. As can be seen, DHAalone stabilizes the level of ccfDNA for up to three days (see donor 1).Particularly stable ccfDNA levels were obtained when using the QGNmixture. Results comparable to the QGN mixture could be obtained whenadding DMAA to the sample in combination with e.g. increasing the EDTAconcentrations and adding a caspase inhibitor.

Example 10 Influence of Sugar Alcohol in Combination with CaspaseInhibitor and Increased EDTA Concentrations on ccfDNA Level

10 ml whole blood samples of two donors were first collected in the BDVacutainer K2E-EDTA (4.45 mM EDTA=reference). Then, 2 ml of thefollowing solutions were added (given concentrations represent finalconcentration in stabilized blood solution):

1: 0.5 M DHA, 1 μM OPH, 14 mM EDTA (QGN mixture);

2: 0.5 M Inositol in QGN mix (without DHA);

3: 0.01M Maltitol in QGN mix (without DHA).

The samples were then processed as described in example 8. The resultsare shown in FIG. 12. The best results were obtained for the QGNmixture. Combinations of sugar alcohol to the QGN mixture also showedstabilizing effects when compared to the EDTA stabilized samples.

Example 11 Influence of Combinations of DMAA and OPH (Caspase Inhibitor)Concentrations on ccfDNA Levels

10 ml whole blood samples were first collected in BD Vacutainer K2E-EDTA(4.45 mM EDTA=reference). Then, 2 ml of the following solutions wereadded (given concentrations represent final concentration in stabilizedblood solution). Each condition was tested with six tubes from differentdonors.

1: EDTA reference (BD Vacutainer K2E);

2: QGN mixture;

3: 50 mg EDTA, 1 μM OPH;

4: 50 mg EDTA, 2 μM OPH;

5: 50 mg EDTA, 1 μM OPH, 5% DMAA;

6: 50 mg EDTA, 1 μM OPH, 10% DMAA;

7: 50 mg EDTA, 2 μM OPH, 5% DMAA;

8: 50 mg EDTA; 2 μM OPH and 10% DMAA.

The sample incubation, isolation of plasma and isolation from nucleicacids from the cleared plasma fraction were performed as described inexample 8. However, after the first centrifugation step at 3.000 rpm,plasma samples of identical stabilization conditions were pooled beforethe second centrifugation step for plasma clearing (16.000×g) wascarried out. The results are shown in FIG. 13. As can be seen, differentDMAA concentrations in combination to different OPH concentrations showcomparable results to the QGN mixture. FIG. 14 shows the influence ofcombinations of DMAA and OPH concentrations on ccfDNA levels (differentscaling due to exclosure of reference data).

Example 12 Influence of Combination of QGN Mixture with Sugar Alcoholson ccfDNA Level

10 ml whole blood samples were collected in BD Vacutainer K2E-EDTA (4.45mM EDTA=reference). Afterwards, 2 ml of the following solutions wasadded (given concentrations represent final concentration in stabilizedblood solution). Each condition was tested with six tubes and thus sixdifferent donors.

1: EDTA reference (BD Vacutainer K2E);

2: QGN mixture (0.01M DHA, 14 mM EDTA, 1 μM OPH);

3: 0.01M DHA;

4: 5% DMAA, 14 mM EDTA, 1 μM OPH;

5: 0.01M DHA, 3% DMAA, 1 μM OPH, 14 mM EDTA;

6: 1 μM OPH, 14 mM EDTA, 0.01M DHA, 0.01M Maltitol;

7: 1 μM OPH, 14 mM EDTA, 0.01M DHA, 0.05 M Inositol;

8: 1 μM OPH, 14 mM EDTA, 0.01M DHA, 0.05M Inositol, 0.01M Maltitol.

The samples were processed as described in example 11. However, thesamples were not stored at room temperature, but at 37° C. instead. Theresults are shown in FIG. 15. As can be seen, stable levels of ccfDNAwere achieved, especially when 5% DMAA was added in combination with 14mM EDTA and 1 μM OPH. Therefore, unexpectedly, a very good stabilizationof ccfDNA in whole blood could be achieved even if at elevatedtemperatures (37° C.).

Example 13 Incubation at 37° C.—Analysis of Single Donor Samples

Whole blood samples from six different donors were collected in BDVacutainers K2E, and then 2 ml of the following stabilization solutionswere added per 10 ml whole blood (given concentrations represent finalconcentration in stabilized blood solution):

1: 2 μM OPH, 14 mM EDTA, 5% DMAA;

2: 1 μM OPH, 14 mM EDTA, 3% DMAA;

3: 1 μM OPH, 14 mM EDTA, 0.01M DHA, 3% DMAA.

The samples were incubated at 37° C. for up to six days. Otherwise, thesame procedure as in example 8 was followed. The results are shown inFIGS. 16 and 17. As can be seen, for all six donors, the level of ccfDNAwas preserved when different concentrations of DMAA in combination withOPH and EDTA were added to the blood samples. Therefore, an efficientstabilization can be achieved with the method according to the presentinvention.

Example 14 Limit of Detection (LoD)

Extracellular nucleic acids are often comprised in very small amounts inthe sample. Therefore, it is important to have a stabilization procedurewhich not only efficiently preserves the extracellular nucleic acidswithin the stabilized sample, but additionally allows to subsequentlyisolate the extracellular nucleic acids with high yield from thestabilized sample. Example 14 demonstrates that the stabilization methodaccording to the present invention is superior to prior artstabilization methods in that the extracellular nucleic acids can beisolated with higher yield from the stabilized samples. Thisadvantageously reduces the limit of detection and thus, allows toreliably determine also rare target nucleic acids within the populationof extracellular nucleic acids.

The following stabilization solutions/tubes were compared:

-   1. Cell-free RNA BCT (Streck Inc, cat. #:218976—comprises a    formaldehyde releaser as stabilizer)-   2. BD Vacutainer K2E (BD, Cat. #: 367525—comprises EDTA)=reference-   3. QGN stabilization (5% DMAA, 14 mM EDTA, 2 μM OPH (caspase    inhibitor))

Whole blood samples were collected in cell-free RNA BCT and BDVacutainer K2E tubes. To one half of blood collected in BD tubes, theQGN stabilization solution was added. Thus, the sample stabilizedaccording to the invention comprise an additional amount of EDTA that iscontributed by the BD Vacutainer stabilization. The samples werecentrifuged at 3.000×rpm for 10 minutes, and the obtained plasma wasaliquoted to 1.5 ml replicates. Afterwards, the following amounts of DNAspike-in control (1.000 bp) were added per sample: 1.000 copies, 5000copies, 100 copies, 50 copies and 10 copies.

8 replicates of 500 to 10 copies/sample, 4 replicates of 1.000copies/sample and 5 copies/sample were prepared. The samples wereincubated for 3 days at room temperature. The sample preparation wasdone on the QIAsymphony SP automated system, using the QIAsymphonyvirus/bacteria Cell-free 1000 application which allows isolatingextracellular nucleic acids from plasma samples. The nucleic acids wereeluted in 60 μl; the subsequent PCR was performed in triplicates.

The results are shown in FIG. 18. As can be seen, 100% hit >1.000 copiesper sample was obtained when using either the BD EDTA tubes or thestabilization solution according to the present invention. This showsthat the isolation of nucleic acids is not impaired when using thestabilization solution according to the present invention. In contrast,the stabilization that is based on the use of a formaldehyde releaser(Streck) shows a strong inhibition of the nucleic acid isolation. As canbe seen, significantly less nucleic acids could be isolated from therespective samples, even with those samples wherein 500 or even 1.000copies were spiked in. Furthermore, FIG. 18 shows that the bestsensitivity was obtained with a sample stabilized according to thepresent invention. Even for those embodiments wherein only 10 copies persample were spiked in, still 13% positive PCR hits were obtained. Thus,the method according to the present invention not only efficientlystabilizes the samples such as blood samples but furthermore allows thesubsequent recovery of even very low-abundant extracellular nucleicacids. This is an important advantage because it makes this methodparticularly suitable for diagnostic applications and e.g. the detectionof rare target extracellular nucleic acids such as e.g. tumor derivedextracellular nucleic acids or fetal nucleic acids. In particular, inthe lower copy numbers, the stabilization solution that is based on theuse of formaldehyde releasers had a very low performance and showed thehighest limit of detection.

This is also confirmed by the following table:

95% confidence Dose for interval DNA centile 95 min max FragmentTube/stabilizing [copies] [copies] [copies] 1000 bp BD K2E 386 230 995Streck RNA 9902 2909 164606 QGN 599 319 1749

As can be seen from said table, for the 1.000 by fragment, the resultsachieved with EDTA sample and the stabilization solution of the presentinvention is comparable. Thus, the stabilization according to theinvention does not impair the subsequent isolation of nucleic acids.Stabilization using a formaldehyde releaser showed the highest limit ofdetection and thus demonstrates that the subsequent isolation of thenucleic acid was strongly impaired. Therefore, the stabilizationaccording to the present invention is suitable for sensitive detectionof rare ccfDNA targets, which is not achieved by using state of the artmethods.

This is also confirmed by the results shown in FIGS. 19 and 20. As canbe seen, comparable ccfDNA yields are obtained for EDTA stabilizedsamples and samples stabilized using the method according to the presentinvention (measured by 18 S rDNA qPCR). However, reduced ccfDNA yieldswere obtained for the stabilization, which involves the use offormaldehyde releasers (Streck tubes). The yield of formaldehydestabilized samples was reduced by approximately 50% compared to the EDTAstabilized samples. In contrast, the stabilization reagent according tothe present invention has no adverse effect on ccfDNA yield, when usingconventional nucleic acid isolation methods. This is an importantadvantage as it allows to integrate the stabilization method accordingto the present invention into existing nucleic acid isolation proceduresand workflows.

Example 15 Spike-In of 10{circumflex over ( )}4 IU/ml HIV, HCV to WholeBlood Samples of 3 Donors

Whole blood samples were collected in BD Vacutainer 2KE tubes.Afterwards 2 ml of the following stabilization solution was added (givenconcentrations represent final concentration in stabilized bloodsolution). Then, HIV and HCV were added to the whole blood samples at10{circumflex over ( )}4 IU/ml.

1: 5% DMAA, 50 mg EDTA, 1 μM OPH, 0.05 M Inositol;

2: 5% DMAA, 50 mg EDTA, 1 μM OPH, 0.01M Maltitol;

3: 5% DMAA, 50 mg EDTA, 1 μM OPH, 0.05 M Inositol; 0.01M Maltitol;

4: 2% Inositol, 4% Sorbitol.

BD Vacutainer K2E stabilized samples served again as reference.

The samples were incubated at room temperature for up to six days at 37°C. On day 0 and day 3, replicates were processed as follows: the sampleswere centrifuged at 3.000 rpm, for 15 minutes at room temperature tocollect the plasma. The obtained plasma was then again centrifuged at16.000×g for 10 minutes, at 4° C. Extracellular nucleic acids obtainedfrom the cleared plasma supernatant was purified using the QIAsymphonyvirus/bacteria Cell-free 1000 protocol. 1 ml plasma was used as inputmaterial, 60 μl volume was used for elution. The results are shown inFIG. 21. As can be seen, combining DMAA, EDTA and OPH with sugaralcohols allows to stabilize viral nucleic acids levels for up to threedays at 37° C. Therefore, the method according to the present inventionis particularly suitable for diagnostic applications and is alsosuitable for stabilizing the samples in environments wherein potentiallyno refrigerating facilities are available. ΔCt between day 0 and day 3is reduced (ΔCt of approximately 2.5 to ΔCt of approximately 1) comparedto the EDTA blood reference. Furthermore, stabilization effects wereseen with a combination of Sorbitol in combination with Inositol (ΔCt ofapproximately 1 to 1.4).

FIG. 22 shows the decrease of HCV in whole blood that was incubated at37° C. Again, it is shown that when combining DMAA, EDTA and OPH withsugar alcohols, the HCV nucleic acid level is stabilized, indicated by aslowed decline in viral RNA levels, for three days at 37° C. ΔCt betweenday 0 and day 3 is reduced (ΔCt of approximately 1) compared to the EDTAblood reference (ΔCt of approximately 2-3). Furthermore, goodstabilizing effects were achieved for Sorbitol in combination withInositol.

Example 16 Stabilization with N,N Dimethylpropanamid and CaspaseInhibitor

Blood from two different donors was collected into 10 ml K2 EDTA tubes(BD). 4.5 ml of the respectively collected blood was mixed with 0.9 mlstabilization solution containing (per ml of stabilization solution):34.2 mg K2 EDTA, 1.2 ml Quinoline-Val-Asp-CH2-OPH (caspase inhibitor)solution (1 mg dissolved in 388 μl DMSO) and 0.15 g or 0.3 g, or 0.45 gN,N dimethylpropanamide or 0.3 ml DMAA, respectively. Thereby, thefollowing final concentration in the blood/stabilization mixture wasobtained which is as follows:

5.7 mg K2 EDTA, 1 μM Quinoline-Val-Asp-CH2-OPH (caspase inhibitor) and2.5, 5 or 7.5% (w/v) NN dimethylpropanamide or 5% (v/v) DMAA,respectively.

All stabilized blood samples were set up in triplicates per conditionand test time point. At time point 0 (reference), immediately aftermixing the stabilization solution and blood, plasma was generated andthe ccfDNA was extracted. The residual blood sample was stored for threedays and six days at room temperature. As a control, the EDTA stabilizedblood sample was also stored for 3 and 6 days. The plasma was generatedfrom the stabilized and unstabilized (EDTA) blood samples by invertingthe blood containing tubes for four times. Then, the tubes werecentrifuged for 15 minutes at 3.000 rpm/1912×g. 2.5 ml of the plasmafraction was transferred into a fresh 15 ml falcon tube and centrifugedfor 10 minutes at 16.000×g. 2 ml of the respectively cleared plasma wasused for isolating the extracellular nucleic acid using the QIAampcirculating nucleic acid kit.

The results are shown in shown in FIGS. 23 and 24. Shown is the increaseof DNA relative to time point 0 with 2.5%, 5% and 7.5% N,Ndimethylpropanamide or 5% DMAA (fold change) using different ampliconlengths of 18SrRNA gene. Bars indicate the mean of the triplicatesamples per condition and test time point. All solutions according tothe present inventions show significantly lower amounts of released DNAafter storage for 3 and 6 days at room temperature compared to theunstabilized EDTA blood.

The invention claimed is:
 1. A container suitable for collecting a biological sample, comprising: a stabilizing composition suitable for stabilizing an extracellular nucleic acid population comprised in the sample, wherein said stabilizing composition comprises (a) a caspase inhibitor; and the container further comprising at least one compound according to formula 1

wherein R1 is a hydrogen residue or an alkyl residue, R2 and R3 are identical or different hydrocarbon residues with a length of the carbon chain of 1-20 atoms arranged in a linear or branched manner, and R4 is an oxygen, sulphur or selenium residue.
 2. The container according to claim 1, wherein the sample is a blood, plasma or serum sample.
 3. The container according to claim 1, wherein the at least one compound according to formula 1 is comprised in the stabilizing composition.
 4. The container according to claim 1, wherein in formula 1, R1 is a C1-C5 alkyl residue.
 5. The container according to claim 1, wherein the stabilizing composition additionally comprises an anticoagulant.
 6. The container according to claim 1, wherein the container is evacuated.
 7. The container according to claim 1, wherein the stabilizing composition is capable of reducing (1) the release of genomic DNA from cells contained in the sample into the cell-free portion of the sample, and/or (2) the degradation of nucleic acids present in the sample.
 8. The container according to claim 1, wherein a) the caspase inhibitor has one or more of the following characteristics: i) it is a pancaspase inhibitor, ii) it is a caspase-specific peptide, iii) it is a caspase-specific peptide modified by an aldehyde, nitrile or ketone compound, and/or iv) it is selected from the group consisting of Q-VD-OPh having the structure:

and Z-Val-Ala-Asp(OMe)-FMK having the structure:

and/or b) wherein the compound according to formula 1 has one or more of the following characteristics: i) R1, R2 and R3 comprise 1 to 5 carbon atoms, ii) R1, R2 and R3 comprise 1 or 2 carbon atoms, iii) R4 is oxygen, iv) it is a N,N-dialkyl-carboxylic acid amide, v) it is selected from the group consisting of N,N-dimethylacetamide, N,N-diethylacetamide, N,N-dimethylformamide and N,N-diethylformamide, and/or vi) it is N,N-dimethylpropanamide.
 9. The container according to claim 1, wherein the stabilizing composition additionally comprises at least one anticoagulant.
 10. The container according to claim 9, wherein the at least one anticoagulant is a chelating agent.
 11. The container according to claim 10, wherein the chelating agent is EDTA.
 12. The container according to claim 1, wherein the stabilizing composition comprises the caspase inhibitor in a concentration that when the sample is added to the stabilizing composition, the resulting mixture has one or both of the following characteristics: a) it comprises the caspase inhibitor in a concentration of at least 0.01 μM, at least 0.05 μM, at least 0.1 μM, at least 0.5 μM, at least 1 μM, at least 2.5 μM or at least 3.5 μM; b) it comprises the caspase inhibitor in a concentration range selected from 0.01 μM to 100 μM, 0.05 μM to 100 μM, 0.1 μM to 50 μM, 1 μM to 40 μM, 1 μM to 30 μM, and 2.5 μM to 25 μM.
 13. The container according to claim 1, wherein the stabilizing composition comprises in addition to the caspase inhibitor: (b) at least one hypertonic agent which stabilizes cells potentially comprised in the sample, (c) the at least one compound according to formula 1; and (d) optionally at least one anticoagulant.
 14. The container according to claim 1, wherein the stabilizing composition comprises the compound according to formula 1 in a concentration that when the sample is added to the stabilizing composition, the resulting mixture has one or both of the following characteristics: a) it comprises the compound according to formula 1 in a concentration of at least 0.1%, at least 0.5%, at least 1%, at least 0.75%, at least 1%, at least 1.25% or at least 1.5%; b) it comprises the compound according to formula 1 in a concentration range selected from 0.1% to 50%, 0.5% to 25%, 0.75% to 20%, 1% to 15%, and 1% to 10%.
 15. The container according to claim 1, wherein the stabilizing composition comprises: (a) at least one pancaspase inhibitor as caspase inhibitor, (b) at least one hypertonic agent, (c) the at least one compound according to formula 1, and (d) optionally a chelating agent as anticoagulant.
 16. The container according to claim 1, wherein stabilization of the extracellular nucleic acid population is achievable without refrigeration for a time period selected from a) at least one day; b) at least two days; c) at least three days; d) one day to three days; e) one day to six days; and/or f) one day to seven days.
 17. The container according to claim 1, wherein the container has an open top, a bottom, and a sidewall extending therebetween defining a chamber, wherein the stabilization composition is comprised in the chamber.
 18. The container according to claim 17, wherein the container is a tube, the bottom is a closed bottom, the container further comprises a closure in the open top, and the chamber is at a reduced pressure.
 19. The container according to claim 18, wherein the closure is capable of being pierced with a needle or cannula, and wherein the reduced pressure is selected to draw a specified volume of a liquid sample into the chamber.
 20. The container of claim 19, wherein the chamber is at a reduced pressure selected to draw a specified volume of a liquid sample into the chamber, and wherein the stabilizing composition is a liquid and is disposed in the chamber such that the volumetric ratio of the stabilising composition to the specified volume of the sample is selected from 10:1 to 1:20, 5:1 to 1:15, 1:1 to 1:10 and 1:2 to 1:5.
 21. The container according to claim 1, suitable for collecting a blood sample, wherein the stabilizing composition further comprises an anticoagulant, and is capable of reducing (1) the release of genomic DNA from cells in the sample into the cell-free portion of the sample, and (2) the degradation of nucleic acids in the sample.
 22. The container according to claim 13, wherein the stabilizing composition comprises the hypertonic agent in a concentration that when the sample is added to the stabilizing composition, the resulting mixture has one or both of the following characteristics: a) it comprises the hypertonic agent in a concentration of at least 0.05M or at least 0.1M; b) it comprises the hypertonic agent in a concentration range selected from 0.05M to 2M, 0.1 to 1.5M, 0.15M to 0.8M, 0.2M to 0.7M, and 0.1M to 0.6M.
 23. The container according to claim 1, wherein the stabilizing composition has one or more of the following characteristics: a) it is capable of reducing the release of genomic DNA from cells contained in the sample into the cell-free portion of the sample; b) it is capable of reducing the degradation of nucleic acids present in the sample; c) it is provided in a solid form; d) it is provided in a liquid form; and/or e) it is capable of stabilizing the extracellular nucleic acid population contained in said sample at room temperature for at least 3 days.
 24. The container according to claim 23, wherein in b), the stabilizing composition is capable of reducing the degradation of genomic DNA present in the sample.
 25. The container according to claim 1, wherein the container additionally comprises the sample, and wherein the sample has one or more of the following characteristics: a) it comprises extracellular nucleic acids; b) it is selected from the group consisting of whole blood, plasma, serum, lymphatic fluid, urine, liquor, cerebrospinal fluid, ascites, milk, stool, bronchial lavage, saliva, amniotic fluid, semen/seminal fluid, swabs/smears, body fluids, body secretions, nasal secretions, vaginal secretions, wound secretions and excretions and cell culture supernatants; c) it is a cell-depleted or cell containing body fluid; d) it is selected from whole blood, plasma and/or serum; and/or e) it is whole blood.
 26. A method for collecting a sample, comprising collecting a sample from a patient into a chamber of the container according to claim
 1. 27. The container according to claim 13, wherein the hypertonic agent has one or more of the following characteristics: i) it is uncharged, ii) it stabilizes the cells comprised in the sample by inducing cell shrinking, iii) it is cell impermeable, iv) it is water-soluble, v) it is a hydroxylated organic compound, vi) it is a polyol, vii) it is a hydroxy-carbonyl compound, viii) it is a carbohydrate or a sugar alcohol, and/or ix) it is dihydroxyacetone. 